Suitable methods of genetic modification (also referred to as “transformation”) include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al. Adv Drug Deliv Rev. 2012 Sep. 13. pii: 50169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023), and the like.

The choice of method of genetic modification is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (e.g., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

A “target nucleic acid” as used herein is a polynucleotide (e.g., DNA such as genomic DNA) that includes a site (“target site” or “target sequence”) targeted by an RNA-guided endonuclease polypeptide (e.g., wild-type Cas12L; variant Cas12L; fusion Cas12L; etc.). The target sequence is the sequence to which the guide sequence of a subject Cas12L guide RNA (e.g., a dual Cas12L guide RNA or a single-molecule Cas12L guide RNA) will hybridize. For example, the target site (or target sequence) 5′-GAGCAUAUC-3′ within a target nucleic acid is targeted by (or is bound by, or hybridizes with, or is complementary to) the sequence 5′-GAUAUGCUC-3′. Suitable hybridization conditions include physiological conditions normally present in a cell. For a double stranded target nucleic acid, the strand of the target nucleic acid that is complementary to and hybridizes with the guide RNA is referred to as the “complementary strand” or “target strand”; while the strand of the target nucleic acid that is complementary to the “target strand” (and is therefore not complementary to the guide RNA) is referred to as the “non-target strand” or “non-complementary strand.”

By “cleavage” it is meant the breakage of the covalent backbone of a target nucleic acid molecule (e.g., RNA, DNA). Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events.

“Nuclease” and “endonuclease” are used interchangeably herein to mean an enzyme which possesses catalytic activity for nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).

By “cleavage domain” or “active domain” or “nuclease domain” of a nuclease it is meant the polypeptide sequence or domain within the nuclease which possesses the catalytic activity for nucleic acid cleavage. A cleavage domain can be contained in a single polypeptide chain or cleavage activity can result from the association of two (or more) polypeptides. A single nuclease domain may consist of more than one isolated stretch of amino acids within a given polypeptide.

The term “stem cell” is used herein to refer to a cell (e.g., plant stem cell, vertebrate stem cell) that has the ability both to self-renew and to generate a differentiated cell type (see Morrison et al. (1997) Cell 88:287-298). In the context of cell ontogeny, the adjective “differentiated”, or “differentiating” is a relative term. A “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with. Thus, pluripotent stem cells (described below) can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells), which in turn can differentiate into cells that are further restricted (e.g., neuron progenitors), which can differentiate into end-stage cells (i.e., terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.), which play a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further. Stem cells may be characterized by both the presence of specific markers (e.g., proteins, RNAs, etc.) and the absence of specific markers. Stem cells may also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny.

Stem cells of interest include pluripotent stem cells (PSCs). The term “pluripotent stem cell” or “PSC” is used herein to mean a stem cell capable of producing all cell types of the organism. Therefore, a PSC can give rise to cells of all germ layers of the organism (e.g., the endoderm, mesoderm, and ectoderm of a vertebrate). Pluripotent cells are capable of forming teratomas and of contributing to ectoderm, mesoderm, or endoderm tissues in a living organism. Pluripotent stem cells of plants are capable of giving rise to all cell types of the plant (e.g., cells of the root, stem, leaves, etc.).

PSCs of animals can be derived in a number of different ways. For example, embryonic stem cells (ESCs) are derived from the inner cell mass of an embryo (Thomson et. al, Science. 1998 Nov 6;282(5391):1145-7) whereas induced pluripotent stem cells (iPSCs) are derived from somatic cells (Takahashi et al., Cell. 2007 Nov. 30;131(5):861-72; Takahashi et al., Nat Protoc. 2007; 2(12):3081-9; Yu et al., Science. 2007 Dec. 21;318(5858):1917-20. Epub 2007 Nov. 20). Because the term PSC refers to pluripotent stem cells regardless of their derivation, the term PSC encompasses the terms ESC and iPSC, as well as the term embryonic germ stem cells (EGSC), which are another example of a PSC. PSCs may be in the form of an established cell line, they may be obtained directly from primary embryonic tissue, or they may be derived from a somatic cell. PSCs can be target cells of the methods described herein.

By “embryonic stem cell” (ESC) is meant a PSC that was isolated from an embryo, typically from the inner cell mass of the blastocyst. ESC lines are listed in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). Stem cells of interest also include embryonic stem cells from other primates, such as Rhesus stem cells and marmoset stem cells. The stem cells may be obtained from any mammalian species, e.g. human, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc. (Thomson et al. (1998) Science 282:1145; Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol. Reprod. 55:254; Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). In culture, ESCs typically grow as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nucleoli. In addition, ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Alkaline Phosphatase, but not SSEA-1. Examples of methods of generating and characterizing ESCs may be found in, for example, U.S. Pat. Nos. 7,029,913, 5,843,780, and 6,200,806, the disclosures of which are incorporated herein by reference. Methods for proliferating hESCs in the undifferentiated form are described in WO 99/20741, WO 01/51616, and WO 03/020920.

By “embryonic germ stem cell” (EGSC) or “embryonic germ cell” or “EG cell” is meant a PSC that is derived from germ cells and/or germ cell progenitors, e.g. primordial germ cells, i.e. those that would become sperm and eggs. Embryonic germ cells (EG cells) are thought to have properties similar to embryonic stem cells as described above. Examples of methods of generating and characterizing EG cells may be found in, for example, U.S. Pat. No. 7,153,684; Matsui, Y., et al., (1992) Cell 70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113; Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U., et al. (1996) Development, 122:1235, the disclosures of which are incorporated herein by reference.

By “induced pluripotent stem cell” or “iPSC” it is meant a PSC that is derived from a cell that is not a PSC (i.e., from a cell this is differentiated relative to a PSC). iPSCs can be derived from multiple different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods of generating and characterizing iPSCs may be found in, for example, U.S. Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, the disclosures of which are incorporated herein by reference. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors (e.g. Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells.

By “somatic cell” it is meant any cell in an organism that, in the absence of experimental manipulation, does not ordinarily give rise to all types of cells in an organism. In other words, somatic cells are cells that have differentiated sufficiently that they will not naturally generate cells of all three germ layers of the body, i.e. ectoderm, mesoderm and endoderm. For example, somatic cells would include both neurons and neural progenitors, the latter of which may be able to naturally give rise to all or some cell types of the central nervous system but cannot give rise to cells of the mesoderm or endoderm lineages.

By “mitotic cell” it is meant a cell undergoing mitosis. Mitosis is the process by which a eukaryotic cell separates the chromosomes in its nucleus into two identical sets in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly equal shares of these cellular components.

By “post-mitotic cell” it is meant a cell that has exited from mitosis, i.e., it is “quiescent”, i.e. it is no longer undergoing divisions. This quiescent state may be temporary, i.e. reversible, or it may be permanent.

By “meiotic cell” it is meant a cell that is undergoing meiosis. Meiosis is the process by which a cell divides its nuclear material for the purpose of producing gametes or spores. Unlike mitosis, in meiosis, the chromosomes undergo a recombination step which shuffles genetic material between chromosomes. Additionally, the outcome of meiosis is four (genetically unique) haploid cells, as compared with the two (genetically identical) diploid cells produced from mitosis.

In some instances, a component (e.g., a nucleic acid component (e.g., a Cas12L guide RNA); a protein component (e.g., wild-type Cas12L polypeptide; variant Cas12L polypeptide; fusion Cas12L polypeptide; etc.); and the like) includes a label moiety. The terms “label”, “detectable label”, or “label moiety” as used herein refer to any moiety that provides for signal detection and may vary widely depending on the particular nature of the assay. Label moieties of interest include both directly detectable labels (direct labels; e.g., a fluorescent label) and indirectly detectable labels (indirect labels; e.g., a binding pair member). A fluorescent label can be any fluorescent label (e.g., a fluorescent dye (e.g., fluorescein, Texas red, rhodamine, ALEXAFLUOR® labels, and the like), a fluorescent protein (e.g., green fluorescent protein (GFP), enhanced GFP (EGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), cherry, tomato, tangerine, and any fluorescent derivative thereof), etc.). Suitable detectable (directly or indirectly) label moieties for use in the methods include any moiety that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means. For example, suitable indirect labels include biotin (a binding pair member), which can be bound by streptavidin (which can itself be directly or indirectly labeled). Labels can also include: a radiolabel (a direct label)(e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P); an enzyme (an indirect label)(e.g., peroxidase, alkaline phosphatase, galactosidase, luciferase, glucose oxidase, and the like); a fluorescent protein (a direct label)(e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and any convenient derivatives thereof); a metal label (a direct label); a colorimetric label; a binding pair member; and the like. By “partner of a binding pair” or “binding pair member” is meant one of a first and a second moiety, wherein the first and the second moiety have a specific binding affinity for each other. Suitable binding pairs include, but are not limited to: antigen/antibodies (for example, digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl, fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, and rhodamine anti-rhodamine), biotin/avidin (or biotin/streptavidin) and calmodulin binding protein (CBP)/calmodulin. Any binding pair member can be suitable for use as an indirectly detectable label moiety.

Any given component, or combination of components can be unlabeled, or can be detectably labeled with a label moiety. In some cases, when two or more components are labeled, they can be labeled with label moieties that are distinguishable from one another.

General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to an individual organism, e.g., a mammal, including, but not limited to, murines, simians, humans, non-human primates, ungulates, felines, canines, bovines, ovines, mammalian farm animals, mammalian sport animals, and mammalian pets.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a Cas12L CRISPR-Cas effector polypeptide” includes a plurality of such polypeptides and reference to “the guide RNA” includes reference to one or more guide RNAs and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides RNA-guided CRISPR-Cas effector proteins (referred to herein as “Cas12L” polypeptides or “CasLambda” polypeptides or “Cas,” polypeptides), nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising: a Cas12L polypeptide of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid, using a Cas12L polypeptide of the present disclosure and a guide RNA.

The present disclosure provides guide RNAs (referred to herein as “Cas12L guide RNAs” or “CasLambda guide RNAs” or “Cas, guide RNAs”) that bind to and provide sequence specificity to the Cas12L proteins; nucleic acids encoding the Cas12L guide RNAs; and modified host cells comprising the Cas12L guide RNAs and/or nucleic acids encoding same. Cas12L guide RNAs are useful in a variety of applications, which are provided.

Compositions

Crispr/Cas12L Proteins and Guide RNAs

A Cas12L CRISPR/Cas effector polypeptide (e.g., a Cas12L protein) interacts with (binds to) a corresponding guide RNA (e.g., a Cas12L guide RNA) to form a ribonucleoprotein (RNP) complex that is targeted to a particular site in a target nucleic acid (e.g. a target DNA) via base pairing between the guide RNA and a target sequence within the target nucleic acid molecule. A guide RNA includes a nucleotide sequence (a guide sequence) that is complementary to a sequence (the target site) of a target nucleic acid. Thus, a Cas12L protein forms a complex with a Cas12L guide RNA and the guide RNA provides sequence specificity to the RNP complex via the guide sequence. The Cas12L protein of the complex provides the site-specific activity. In other words, the Cas12L protein is guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence (e.g. a chromosomal sequence or an extrachromosomal sequence, e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) by virtue of its association with the guide RNA.

The present disclosure provides compositions comprising a Cas12L polypeptide (and/or a nucleic acid comprising a nucleotide sequence encoding the Cas12L polypeptide) (e.g., where the Cas12L polypeptide can be a naturally existing protein, a nickase Cas12L protein, a catalytically inactive (“dead” Cas12L; also referred to herein as a “dCas12L protein”), a fusion Cas12L protein, etc.). The present disclosure provides compositions comprising a Cas12L guide RNA (and/or a nucleic acid comprising a nucleotide sequence encoding the Cas12L guide RNA). The present disclosure provides compositions comprising (a) a Cas12L polypeptide (and/or a nucleic acid encoding the Cas12L polypeptide) (e.g., where the Cas12L polypeptide can be a naturally existing protein, a nickase Cas12L protein, a dCas12L protein, a fusion Cas12L protein, etc.) and (b) a Cas12L guide RNA (and/or a nucleic acid encoding the Cas12L guide RNA). The present disclosure provides a nucleic acid/protein complex (RNP complex) comprising: (a) a Cas12L polypeptide of the present disclosure (e.g., where the Cas12L polypeptide can be a naturally existing protein, a nickase Cas12L protein, a dead Cas12L protein, a fusion Cas12L protein, etc.); and (b) a Cas12L guide RNA.

Cas12L Protein

A Cas12L polypeptide (this term is used interchangeably with the term “Cas12L protein”) can bind and/or modify (e.g., cleave, nick, methylate, demethylate, etc.) a target nucleic acid and/or a polypeptide associated with target nucleic acid (e.g., methylation or acetylation of a histone tail) (e.g., in some cases, the Cas12L protein includes a fusion partner with an activity, and in some cases, the Cas12L protein provides nuclease activity). In some cases, the Cas12L protein is a naturally-occurring protein (e.g., naturally occurs in bacteriophage). In other cases, the Cas12L protein is not a naturally-occurring polypeptide (e.g., the Cas12L protein is a variant Cas12L protein, a fusion Cas12L protein, and the like).

Assays to determine whether given protein interacts with a Cas12L guide RNA can be any convenient binding assay that tests for binding between a protein and a nucleic acid. Suitable binding assays (e.g., gel shift assays) will be known to one of ordinary skill in the art (e.g., assays that include adding a Cas12L guide RNA and a protein to a target nucleic acid). Assays to determine whether a protein has an activity (e.g., to determine if the protein has nuclease activity that cleaves a target nucleic acid and/or some heterologous activity) can be any convenient assay (e.g., any convenient nucleic acid cleavage assay that tests for nucleic acid cleavage). Suitable assays (e.g., cleavage assays) will be known to one of ordinary skill in the art.

A naturally occurring Cas12L protein functions as an endonuclease that catalyzes a double strand break at a specific sequence in a targeted double stranded DNA (dsDNA). The sequence specificity is provided by the associated guide RNA, which hybridizes to a target sequence within the target DNA. The naturally occurring Cas12L guide RNA is a crRNA, where the crRNA includes (i) a guide sequence that hybridizes to a target sequence in the target DNA and (ii) a protein binding segment which includes a stem-loop (hairpin—dsRNA duplex) that binds to the Cas12L protein.

In some embodiments, the Cas12L protein of the subject methods and/or compositions is (or is derived from) a naturally occurring (wild type) protein. Examples of naturally occurring Cas12L proteins are depicted in FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF. In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with any one of the Cas12L amino acid sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF). In some cases, a FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF protein (of the subject compositions and/or methods) includes an amino acid sequence depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF).

In some cases, a Cas12L protein (of the subject compositions and/or methods) has more sequence identity to an amino acid sequence depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF) than to any of the following: Cas12a proteins, Cas12b proteins, Cas12c proteins, Cas12d proteins, Cas12e proteins, Cas12 g proteins, Cas12h proteins, and Cas12i proteins. In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having a RuvC domain (which includes the RuvC-I, RuvC-II, and RuvC-III domains) that has more sequence identity to the RuvC domain of an amino acid sequence depicted in FIG. 2 (e.g., the RuvC domain of any of the Cas12L amino acid sequences depicted in FIG. 2) than to the RuvC domain of any of the following: Cas12a proteins, Cas12b proteins, Cas12c proteins, Cas12d proteins, Cas12e proteins, Cas12 g proteins, Cas12h proteins, and Cas12i proteins.

FIG. 3A-3B provides the locations of active site residues present in RuvC domains of various Cas12L polypeptides depicted in FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF. For example, active site residues of Cas12L_1_257905508 (FIG. 2A) are amino acid residues 336, 530, and 682.

FIG. 5A-5P provide an amino acid sequence alignment of the 58 Cas12L polypeptides depicted in FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF. As shown in FIG. 5A-5P, a number of amino acids are conserved among the 58 Cas12L polypeptides. For example, K14, T15, E37, Y41, Y42, N43, S46, 149, 157, Y73, Y88, F91, N119, L121, N140, Y141, Q169, 1171, E173, W185, Y191, Y205, L209, F212, Y213, F235, G236, G237, C238, R240, G281, N309, K324, 1335, T346, Y357, N359, 1360, Y361, V364, F370, F391, P393, L394, E395, L399, S401, R402, Q406, E417, V423, L424, Y442, K448, L449, R450, K454, A455, V459, K460, Y470, D471, E476, E477, and G594, based on the amino acid numbering of Cas12L_1_257905508 (FIG. 2A) are conserved. Thus, e.g., a Cas12L polypeptide can comprise one or more of K14, T15, E37, Y41, Y42, N43, S46, 149, 157, Y73, Y88, F91, N119, L121, N140, Y141, Q169, 1171, E173, W185, Y191, Y205, L209, F212, Y213, F235, G236, G237, C238, R240, G281, N309, K324, 1335, T346, Y357, N359, 1360, Y361, V364, F370, F391, P393, L394, E395, L399, S401, R402, Q406, E417, V423, L424, Y442, K448, L449, R450, K454, A455, V459, K460, Y470, D471, E476, E477, and G594, based on the amino acid numbering of Cas12L_1_257905508 (FIG. 2A), or the same amino acid at a corresponding position in another Cas12L polypeptide (e.g., a Cas12L polypeptide depicted in FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF), where those skilled in the art would understand, based on the alignment provided in FIG. 5A-5P, what a “corresponding position” would be. A Cas12L polypeptide can comprise a conservative amino acid substitution at one or more of K14, T15, E37, Y41, Y42, N43, S46, I49, I57, Y73, Y88, F91, N119, L121, N140, Y141, Q169, 1171, E173, W185, Y191, Y205, L209, F212, Y213, F235, G236, G237, C238, R240, G281, N309, K324, 1335, T346, Y357, N359, 1360, Y361, V364, F370, F391, P393, L394, E395, L399, S401, R402, Q406, E417, V423, L424, Y442, K448, L449, R450, K454, A455, V459, K460, Y470, D471, E476, E477, and G594, based on the amino acid numbering of Cas12L_1_257905508 (FIG. 2A), or at a corresponding position in another Cas12L polypeptide (e.g., a Cas12L polypeptide depicted in FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF).

FIG. 6A-6C provide an alignment of the amino acid sequences of Cas12L_2_196848753 (FIG. 2B), Cas12L_29_255238293 (FIG. 2BB; referred to as Cas12L_28_255238293 in FIG. 2BB), Cas12L_38_73472625 (FIG. 2KK; referred to as Cas12L_37_73472625 in FIG. 2KK), Cas12L_42_74408273_partial (FIG. 2NN; referred to as Cas12L_41_74408273_partial in FIG. 2NN), Cas12L_54_75257103_partial (FIG. 2PP; referred to as Cas12L_43_75257103_partial in FIG. 2PP), Cas12L_52_82983331 (FIG. 2XX; referred to as Cas12L_51_82983331 in FIG. 2XX), and Cas12L_56_77738117 (FIG. 2BBB; referred to as Cas12L_55_77738117 in FIG. 2BBB. Amino acids in boxes are conserved among the Cas12L polypeptides in FIG. 6A-6C. A Cas12L polypeptide can comprise the amino acids set out in the boxes in FIG. 6A-6C, or a conservative amino acid substitution of such amino acids, at the positions depicted in FIG. 6A-6C.

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the RuvC domain (which includes the RuvC-I, RuvC-II, and RuvC-III domains) of any one of the Cas12L amino acid sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF). In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 70% or more sequence identity (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the RuvC domain (which includes the RuvC-I, RuvC-II, and RuvC-III domains) of any one of the Cas12L amino acid sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF). In some cases, a Cas12L protein (of the subject compositions and/or methods) includes the RuvC domain (which includes the RuvC-I, RuvC-II, and RuvC-III domains) of any one of the Cas12L amino acid sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF).

In some cases, a guide RNA that binds a Cas12L polypeptide includes a nucleotide sequence depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF; where “T” is replaced with “U”) (or in some cases the reverse complement of same). In some cases, the guide RNA comprises the nucleotide sequence (N)nX or the reverse complement of same, where N is any nucleotide, n is an integer from 15 to 30 (e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30), and X is any one of the nucleotide sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIG. 2AAA-2FFF) (or in some cases the reverse complement of same).

An alignment of nucleotide sequences of the repeat region of various CasLambda (Casλ; Cas12L) guide RNAs is provided in FIG. 7. As shown in FIG. 7, CRISPR repeat sequences contain conserved sequence motifs across homologs. In some cases, a guide RNA that binds a Cas12L polypeptide includes a nucleotide sequence (a repeat sequence; or protein-binding sequence) of the following consensus sequence: WAUUGUUGUARMWNYYWUUUURUAWGGWKURAACAAC (SEQ ID NO:162), where W is A or U; R is G or A; M is A or C; N is A, G, C, or U; Y is U or C; and K is G or U. For example, a guide RNA that binds a CasLambda28 polypeptide (Cas12L 28; FIG. 2BB) can comprise a protein-binding segment comprising the nucleotide sequence: AUUGUUGAAAUAGUACUUUUAUAGUCUAUAUACAAC (SEQ ID NO:63). As another example a guide RNA that binds a CasLambda37 polypeptide (Cas12L 37; FIG. 2KK) can comprise a protein-binding segment comprising the nucleotide sequence: AUUGUUGUAACAUCUAUUUUGUAAGGUGUAAACAAC (SEQ ID NO:70). As another example a guide RNA that binds a CasLambda29 polypeptide (Cas12L 29; FIG. 2CC) can comprise a protein-binding segment comprising the nucleotide sequence: UAUUGUUGUAACUCUUAUUUUGUAUGGAGUAAACAAC (SEQ ID NO:64). As another example a guide RNA that binds a CasLambda42 polypeptide (Cas12L 42; FIG. 00) can comprise a protein-binding segment comprising the nucleotide sequence: AAUUGUUGUAACUCUUAUUUUGUAUGGAGUAAACAAC (SEQ ID NO:163). As another example a guide RNA that binds any one of CasLambda polypeptides 1, 3, 4, 5, 7-15, 18, 19, 21-27, 29-35, 38, 40-46, 48, 49, 53, and 54 (any one of Cas12L 1, 3, 4, 5, 7-15, 18, 19, 21-27, 29-35, 38, 40-46, 48, 49, 53, and 54; FIGS. 2A, 2C, 2D, 2E, 2G-2O, 2R, 2S, 2U-2AA, 2CC-2II, 2LL, 2MM-2SS, 2UU, 2VV, 2ZZ, and 2AAA, respectively) can comprise a protein-binding segment comprising the nucleotide sequence: AUUGUUGUAACUCUUAUUUUGUAUGGAGUAAACAAC (SEQ ID NO:59). As another example a guide RNA that binds a CasLambda47 polypeptide (Cas12L 47; FIG. 2TT) can comprise a protein-binding segment comprising the nucleotide sequence: AUUGUUGUAACUUUUAUUUUGUAUGGAGUAAACAAC (SEQ ID NO:65). As another example a guide RNA that binds a CasLambda2 polypeptide (Cas12L 2; FIG. 2B) can comprise a protein-binding segment comprising the nucleotide sequence: AUUGUUGUAGACCUCUUUUUAUAAGGAUUGAACAAC (SEQ ID NO:60). A Cas12L polypeptide of the present disclosure can form a complex (a ribonucleoprotein (RNP) complex) with a guide RNA comprising a protein-binding segment depicted in FIG. 7. In some cases, a Cas12L polypeptide of the present disclosure can form an RNP complex with different guide RNAs comprising a protein-binding segment of different sequences depicted in FIG. 7.

As another example a guide RNA that binds a CasLambda20 polypeptide or a CasLambda52 polypeptide (Cas12L 20 or Cas12L 52; FIGS. 2T and 2YY, respectively) can comprise a protein-binding segment comprising the nucleotide sequence: AAUGUUGUAGAUGCCUUUUUAUAAGGAUUAAACAAC (SEQ ID NO:68). As another example a guide RNA that binds a CasLambda50 polypeptide (Cas12L 50; FIG. 2WW) can comprise a protein-binding segment comprising the nucleotide sequence: AAUGUUGUAGAUACCUUUUUGUAAGGAUUGAACAAC (SEQ ID NO:66). As another example a guide RNA that binds a CasLambda16 polypeptide (Cas12L 16; FIG. 2P) can comprise a protein-binding segment comprising the nucleotide sequence: UAUUGUUGUAGAUACCUUUUUGUAAGGAUUAAACAAC (SEQ ID NO:61). As another example a guide RNA that binds a CasLambda6 polypeptide (Cas12L 6; FIG. 2F) can comprise a protein-binding segment comprising the nucleotide sequence: AUUGUUGUAGAUACCUUUUUGUAAGGAUUGAACAAC (SEQ ID NO:164). As another example a guide RNA that binds a CasLambda51 polypeptide (Cas12L 51; FIG. 2XX) can comprise a protein-binding segment comprising the nucleotide sequence: AUUGUUGUAAUACUAUUUUUGUAAAGUAUAAACAAC (SEQ ID NO:67). As another example a guide RNA that binds a CasLambda55 polypeptide (Cas12L 55; FIG. 2BBB) can comprise a protein-binding segment comprising the nucleotide sequence:

(SEQ ID NO: 69)

AUUGUUGUAAUACACUUUUUAUAAGGUAUGAACAAC.

In addition to containing conserved sequence motifs in the repeat (protein-binding) regions, the repeat region of a CasLambda guide RNA share conserved secondary structures across homologs. FIG. 8A-8M depict the secondary structures of the repeat regions of various CasLambda guide RNAs. For example, the repeat region can include palindromic regions that can form stem and stem-loop structures.

In some cases, a guide RNA that binds a Cas12L polypeptide includes a nucleotide sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with any one of the sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF) (or in some cases the reverse complement of same). In some cases, the guide RNA comprises the nucleotide sequence (N)nX or the reverse complement of same, where N is any nucleotide, n is an integer from 15 to 30 (e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30), and X is a nucleotide sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with any one of the sequences depicted in FIG. 2 (e.g., any one of FIG. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF).

In some cases, a guide RNA that binds a Cas12L polypeptide includes a nucleotide sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with any one of the sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF) (or in some cases the reverse complement of same). In some cases, the guide RNA comprises the nucleotide sequence (N)nX or the reverse complement of same, where N is any nucleotide, n is an integer from 15 to 30 (e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30), and X is a nucleotide sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with any one of the sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF).

In some cases, a guide RNA that binds a Cas12L polypeptide includes a nucleotide sequence depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF) (or in some cases the reverse complement of same). In some cases, the guide RNA comprises the nucleotide sequence X(N)n, where N is any nucleotide, n is an integer from 15 to 30 (e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30), and X is any one of the nucleotide sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF) (or in some cases the reverse complement of same).

In some cases, a guide RNA that binds a Cas12L polypeptide includes a nucleotide sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with any one of the sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF) (or in some cases the reverse complement of same). In some cases, the guide RNA comprises the nucleotide sequence X(N)n, where N is any nucleotide, n is an integer from 15 to 30 (e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30), and X is a nucleotide sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with any one of the sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF).

In some cases, a guide RNA that binds a Cas12L polypeptide includes a nucleotide sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with any one of the sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF) (or in some cases the reverse complement of same). In some cases, the guide RNA comprises the nucleotide sequence X(N)n, where N is any nucleotide, n is an integer from 15 to 30 (e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30), and X is a nucleotide sequence having 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with any one of the sequences depicted in FIG. 2 (e.g., any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF).

FIG. 2A

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2A and designated “Cas12L_1_257905508.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2A. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2A. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2A. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2A. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2A, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2A) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2B

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2B and designated “Cas12L_2_196848753.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2B. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2B. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2B. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2B. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2B, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 675 amino acids (aa) to 800 aa, e.g., from 675 aa to 700 aa, from 700 aa to 725 aa, from 725 aa to 735 aa, from 735 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 735 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2B) includes the following nucleotide sequence: ATTGTTGTAGACCTCTTTTTATAAGGATTGAACAAC (SEQ ID NO:3) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAGACCTCTTTTTATAAGGATTGAACAAC (SEQ ID NO:4) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2C

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2C and designated “Cas12L_3_66741167.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2C. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2C. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2C. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2C. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2C, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 725 amino acids (aa) to 875 aa, e.g., from 725 aa to 750 aa, from 750 aa to 778 aa, from 778 aa to 800 aa, from 800 aa to 825 aa, from 825 aa to 850 aa, or from 850 aa to 875 aa). In some cases, the Cas12L polypeptide has a length of 778 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2C) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2D

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2D and designated “Cas12L_4_67031163.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2D. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2D. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2D. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2D. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2D, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 725 amino acids (aa) to 875 aa, e.g., from 725 aa to 750 aa, from 750 aa to 778 aa, from 778 aa to 800 aa, from 800 aa to 825 aa, from 825 aa to 850 aa, or from 850 aa to 875 aa). In some cases, the Cas12L polypeptide has a length of 778 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2D) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2E

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2E and designated “Cas12L_5_67793351.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2E. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2E. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2E. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2E. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2E, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 725 amino acids (aa) to 875 aa, e.g., from 725 aa to 750 aa, from 750 aa to 778 aa, from 778 aa to 800 aa, from 800 aa to 825 aa, from 825 aa to 850 aa, or from 850 aa to 875 aa). In some cases, the Cas12L polypeptide has a length of 778 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2E) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2F

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 350 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2F and designated “Cas12L_6_67912869_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 350 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2F. In some cases, a Cas12L protein includes a contiguous stretch of about 350 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2F. In some cases, a Cas12L protein includes a contiguous stretch of about 350 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2F. In some cases, a Cas12L protein includes a contiguous stretch of about 350 amino acids comprising the Cas12L amino acid sequence depicted in FIG. 2F. In some cases, a Cas12L protein includes a contiguous stretch of about 350 amino acids having the Cas12L protein sequence depicted in FIG. 2F, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, or from 750 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from about 725 amino acids to about 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2F) includes the following nucleotide sequence: ATTGTTGTAGATACCTTTTTGTAAGGATTGAACAAC (SEQ ID NO:5) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAGATACCTTTTTGTAAGGATTGAACAAC (SEQ ID NO:6) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2G

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2G and designated “Cas12L_7_68090316_partial.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2G. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2G. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2G. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2G. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2G, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 650 amino acids (aa) to 800 aa, e.g., from 650 aa to 700 aa, from 700 aa to 717 aa, from 717 aa to 725 aa, from 725 aa to 750 aa, or from 750 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from about 717 amino acids to about 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2G) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2H

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2H and designated “Cas12L_8_68328292_partial.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2H. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2H. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2H. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2H. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2H, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 742 aa, from 742 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from about 742 amino acids to about 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2H) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2I

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2I and designated “Cas12L_9_68454124.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2I. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2I. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2I. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2I. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2I, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2I) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2J

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2J and designated “Cas12L_10_68605313.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2J. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2J. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2J. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2J. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2J, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 725 amino acids (aa) to 825 aa, e.g., from 725 aa to 750 aa, from 750 aa to 775 aa, from 775 aa to 782 aa, from 782 aa to 800 aa, or from 800 aa to 825 aa). In some cases, the Cas12L polypeptide has a length of 782 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2J) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2K

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 92 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2K and designated “Cas12L_11_69266821_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 92 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2K. In some cases, a Cas12L protein includes a contiguous stretch of about 92 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2K. In some cases, a Cas12L protein includes a contiguous stretch of about 92 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2K. In some cases, a Cas12L protein includes a contiguous stretch of about 92 amino acids having the Cas12L amino acid sequence depicted in FIG. 2K. In some cases, a Cas12L protein includes a contiguous stretch of about 92 amino acids having the Cas12L protein sequence depicted in FIG. 2K, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2K) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2L

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 92 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2L and designated “Cas12L_12_69417229_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 92 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2L. In some cases, a Cas12L protein includes a contiguous stretch of about 92 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2L. In some cases, a Cas12L protein includes a contiguous stretch of about 92 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2L. In some cases, a Cas12L protein includes a contiguous stretch of about 92 amino acids having the Cas12L amino acid sequence depicted in FIG. 2L. In some cases, a Cas12L protein includes a contiguous stretch of about 92 amino acids having the Cas12L protein sequence depicted in FIG. 2L, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2L) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2M

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2M and designated “Cas12L_13_69733214.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2M. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2M. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2M. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2M. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2M, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2M) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2N

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 427 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2N and designated “Cas12L_14_70235246_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 427 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2N. In some cases, a Cas12L protein includes a contiguous stretch of about 427 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2N. In some cases, a Cas12L protein includes a contiguous stretch of about 427 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2N. In some cases, a Cas12L protein includes a contiguous stretch of about 427 amino acids having the Cas12L amino acid sequence depicted in FIG. 2N. In some cases, a Cas12L protein includes a contiguous stretch of about 427 amino acids having the Cas12L protein sequence depicted in FIG. 2N, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 425 amino acids (aa) to 800 aa, e.g., from 425 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to 700 aa, from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2N) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2O

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2O and designated “Cas12L_15_70724743.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2O. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2O. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2O. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2O. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2O, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2O) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2P

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2P and designated “Cas12L_16_70731038.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2P. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2P. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2P. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2P. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2P, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 675 amino acids (aa) to 800 aa, e.g., from 675 aa to 700 aa, from 700 aa to 725 aa, from 725 aa to 735 aa, from 735 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 735 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2P) includes the following nucleotide sequence: TATTGTTGTAGATACCTTTTTGTAAGGATTAAACAAC (SEQ ID NO:7) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nTATTGTTGTAGATACCTTTTTGTAAGGATTAAACAAC (SEQ ID NO:8) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2Q

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2Q and designated “Cas12L_17_70959391.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2Q. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2Q. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2Q. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2Q. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2Q, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids.

FIG. 2R

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2R and designated “Cas12L_18_71078086.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2R. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2R. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2R. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2R. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2R, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2R) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2S

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 680 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2S and designated “Cas12L_19_71193509_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 680 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2S. In some cases, a Cas12L protein includes a contiguous stretch of about 680 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2S. In some cases, a Cas12L protein includes a contiguous stretch of about 680 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2S. In some cases, a Cas12L protein includes a contiguous stretch of about 680 amino acids having the Cas12L amino acid sequence depicted in FIG. 2S. In some cases, a Cas12L protein includes a contiguous stretch of about 680 amino acids having the Cas12L protein sequence depicted in FIG. 2S, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, or from 750 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from about 725 amino acids to about 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2S) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2T

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 516 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2T and designated “Cas12L_20_71210958_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 516 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2T. In some cases, a Cas12L protein includes a contiguous stretch of about 516 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2T. In some cases, a Cas12L protein includes a contiguous stretch of about 516 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2T. In some cases, a Cas12L protein includes a contiguous stretch of about 516 amino acids having the Cas12L amino acid sequence depicted in FIG. 2T. In some cases, a Cas12L protein includes a contiguous stretch of about 516 amino acids having the Cas12L protein sequence depicted in FIG. 2T, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, or from 750 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from about 725 amino acids to about 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2T) includes the following nucleotide sequence: AATGTTGTAGATGCCTTTTTATAAGGATTAAACAACTTG (SEQ ID NO:9) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nAATGTTGTAGATGCCTTTTTATAAGGATTAAACAACTTG (SEQ ID NO:10) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2U

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 585 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2U and designated “Cas12L_21_71317321_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 585 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2U. In some cases, a Cas12L protein includes a contiguous stretch of about 585 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2U. In some cases, a Cas12L protein includes a contiguous stretch of about 585 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2U. In some cases, a Cas12L protein includes a contiguous stretch of about 585 amino acids having the Cas12L amino acid sequence depicted in FIG. 2U. In some cases, a Cas12L protein includes a contiguous stretch of about 585 amino acids having the Cas12L protein sequence depicted in FIG. 2U, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, or from 750 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from about 725 amino acids to about 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2U) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2V

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2V and designated “Cas12L_22_71456687.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2V. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2V. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2V. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2V. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2V, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2V) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2W

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2W and designated “Cas12L_23_71708971.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2W. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2W. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2W. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2W. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2W, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 500 amino acids (aa) to 800 aa, e.g., from 500 aa to 550 aa, from 550 aa to 585 aa, from 585 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to 700 aa, from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 585 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2W) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2X

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2X and designated “Cas12L_24_46035167_partial.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2X. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2X. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2X. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2X. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2X, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 758 aa, from 758 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 758 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2X) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2Y

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 596 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2Y and designated “Cas12L_25_46784254_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 596 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2Y. In some cases, a Cas12L protein includes a contiguous stretch of about 596 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2Y. In some cases, a Cas12L protein includes a contiguous stretch of about 596 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2Y. In some cases, a Cas12L protein includes a contiguous stretch of about 596 amino acids having the Cas12L amino acid sequence depicted in FIG. 2Y. In some cases, a Cas12L protein includes a contiguous stretch of about 596 amino acids having the Cas12L protein sequence depicted in FIG. 2Y, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, or from 750 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from about 725 amino acids to about 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2Y) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2Z

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2Z and designated “Cas12L_26_46464451.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2Z. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2Z. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2Z. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2Z. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2Z, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 625 amino acids (aa) to 800 aa, e.g., from 625 aa to 640 aa, from 640 aa to 650 aa, from 650 aa to 700 aa, from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 640 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2Z) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2AA

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2AA and designated “Cas12L_27_254489164.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2AA. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2AA. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2AA. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2AA. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2AA, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2AA) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2BB

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2BB and designated “Cas12L_28_255238293.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2BB. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2BB. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2BB. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2BB. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2BB, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 759 aa, from 759 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 759 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2BB) includes the following nucleotide sequence: ATTGTTGAAATAGTACTTTTATAGTCTATATACAAC (SEQ ID NO:11) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGAAATAGTACTTTTATAGTCTATATACAAC (SEQ ID NO:12) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2CC

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2CC and designated “Cas12L_29_72167294.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2CC. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2CC. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2CC. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2CC. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2CC, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2CC) includes the following nucleotide sequence: TATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:13) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nTATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:14) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2DD

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2DD and designated “Cas12L_30_72369269.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2DD. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2DD. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2DD. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2DD. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2DD, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2DD) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2EE

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2EE and designated “Cas12L_31_72503976.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2EE. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2EE. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2EE. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2EE. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2EE, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2EE) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2FF

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 47 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2FF and designated “Cas12L_32_72547654_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 47 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2FF. In some cases, a Cas12L protein includes a contiguous stretch of about 47 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2FF. In some cases, a Cas12L protein includes a contiguous stretch of about 47 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2FF. In some cases, a Cas12L protein includes a contiguous stretch of about 47 amino acids having the Cas12L amino acid sequence depicted in FIG. 2FF. In some cases, a Cas12L protein includes a contiguous stretch of about 47 amino acids having the Cas12L protein sequence depicted in FIG. 2FF, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2FF) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2GG

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2GG and designated “Cas12L_33_72907394.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2GG. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2GG. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2GG. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2GG. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2GG, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2GG) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2HH

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 245 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2HH and designated “Cas12L_34_73124743_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 245 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2HH. In some cases, a Cas12L protein includes a contiguous stretch of about 245 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2HH. In some cases, a Cas12L protein includes a contiguous stretch of about 245 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2HH. In some cases, a Cas12L protein includes a contiguous stretch of about 245 amino acids having the Cas12L amino acid sequence depicted in FIG. 2HH. In some cases, a Cas12L protein includes a contiguous stretch of about 245 amino acids having the Cas12L protein sequence depicted in FIG. 2HH, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2HH) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2II

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 178 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2II and designated “Cas12L_35_73503649_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 178 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2II. In some cases, a Cas12L protein includes a contiguous stretch of about 178 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2II. In some cases, a Cas12L protein includes a contiguous stretch of about 178 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2II. In some cases, a Cas12L protein includes a contiguous stretch of about 178 amino acids having the Cas12L amino acid sequence depicted in FIG. 2II. In some cases, a Cas12L protein includes a contiguous stretch of about 178 amino acids having the Cas12L protein sequence depicted in FIG. 2II, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2II) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2JJ

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 85 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2JJ and designated “Cas12L_36_73503649_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 85 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2JJ. In some cases, a Cas12L protein includes a contiguous stretch of about 85 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2JJ. In some cases, a Cas12L protein includes a contiguous stretch of about 85 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2JJ. In some cases, a Cas12L protein includes a contiguous stretch of about 85 amino acids having the Cas12L amino acid sequence depicted in FIG. 2JJ. In some cases, a Cas12L protein includes a contiguous stretch of about 85 amino acids having the Cas12L protein sequence depicted in FIG. 2JJ, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids.

FIG. 2KK

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2KK and designated “Cas12L_37_73472625.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2KK. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2KK. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2KK. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2KK. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2KK, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 767 aa, from 767 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 767 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2KK) includes the following nucleotide sequence: ATTGTTGTAACATCTATTTTGTAAGGTGTAAACAAC (SEQ ID NO:15) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACATCTATTTTGTAAGGTGTAAACAAC (SEQ ID NO:16) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2LL

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 652 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2LL and designated “Cas12L_38_73764039_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 652 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2LL. In some cases, a Cas12L protein includes a contiguous stretch of about 652 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2LL. In some cases, a Cas12L protein includes a contiguous stretch of about 652 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2LL. In some cases, a Cas12L protein includes a contiguous stretch of about 652 amino acids having the Cas12L amino acid sequence depicted in FIG. 2LL. In some cases, a Cas12L protein includes a contiguous stretch of about 652 amino acids having the Cas12L protein sequence depicted in FIG. 2LL, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2LL) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2MM

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2MM and designated “Cas12L_40_74037305.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2MM. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2MM. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2MM. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2MM. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2MM, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 782 aa, or from 782 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 782 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2MM) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2NN

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2NN and designated “Cas12L_41_74408273_partial.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2NN. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2NN. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2NN. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2NN. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2NN, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 850 amino acids (aa) to 950 aa, e.g., from 850 aa to 875 aa, from 875 aa to 889 aa, from 889 aa to 900 aa, or from 900 aa to 950 aa). In some cases, the Cas12L polypeptide has a length of 889 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2NN) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2OO

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 223 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2OO and designated “Cas12L_42_75186079_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 223 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2OO. In some cases, a Cas12L protein includes a contiguous stretch of about 223 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2OO. In some cases, a Cas12L protein includes a contiguous stretch of about 223 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2OO. In some cases, a Cas12L protein includes a contiguous stretch of about 223 amino acids having the Cas12L amino acid sequence depicted in FIG. 2OO. In some cases, a Cas12L protein includes a contiguous stretch of about 223 amino acids having the Cas12L protein sequence depicted in FIG. 2OO, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2OO) includes the following nucleotide sequence: AATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:17) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nAATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:18) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2PP

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 439 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2PP and designated “Cas12L_43_75257103_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 439 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2PP. In some cases, a Cas12L protein includes a contiguous stretch of about 439 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2PP. In some cases, a Cas12L protein includes a contiguous stretch of about 439 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2PP. In some cases, a Cas12L protein includes a contiguous stretch of about 439 amino acids having the Cas12L amino acid sequence depicted in FIG. 2PP. In some cases, a Cas12L protein includes a contiguous stretch of about 439 amino acids having the Cas12L protein sequence depicted in FIG. 2PP, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2PP) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2QQ

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 196 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2QQ and designated “Cas12L_44_75257103_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 196 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2QQ. In some cases, a Cas12L protein includes a contiguous stretch of about 196 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2QQ. In some cases, a Cas12L protein includes a contiguous stretch of about 196 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2QQ. In some cases, a Cas12L protein includes a contiguous stretch of about 196 amino acids having the Cas12L amino acid sequence depicted in FIG. 2QQ. In some cases, a Cas12L protein includes a contiguous stretch of about 196 amino acids having the Cas12L protein sequence depicted in FIG. 2QQ, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2QQ) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2RR

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2RR and designated “Cas12L_45_75616607_partial.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2RR. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2RR. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2RR. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2RR. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2RR, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 748 aa, from 748 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 748 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2RR) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2SS

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 481 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2SS and designated “Cas12L_46_75784289_partial.” For example, in some cases, a Cas12L) includes a contiguous stretch of about 481 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2SS. In some cases, a Cas12L protein) includes a contiguous stretch of about 481 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2SS. In some cases, a Cas12L protein) includes a contiguous stretch of about 481 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2SS. In some cases, a Cas12L protein) includes a contiguous stretch of about 481 amino acids having the Cas12L amino acid sequence depicted in FIG. 2SS. In some cases, a Cas12L protein) includes a contiguous stretch of about 481 amino acids having the Cas12L protein sequence depicted in FIG. 2SS, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2SS) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2TT

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2TT and designated “Cas12L_47_76512228.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2TT. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2TT. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2TT. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2TT. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2TT, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2TT) includes the following nucleotide sequence: ATTGTTGTAACTTTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:19) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTTTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:20) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2UU

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2UU and designated “Cas12L_48_76600450.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2UU. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2UU. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2UU. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2UU. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2UU, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2UU) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2VV

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2VV and designated “Cas12L_49_44880081.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2VV. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2VV. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2VV. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2VV. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2VV, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2VV) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2WW

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2WW and designated “Cas12L_50_83012613.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2WW. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2WW. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2WW. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2WW. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2WW, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 735 aa, from 735 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 735 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2WW) includes the following nucleotide sequence: AATGTTGTAGATACCTTTTTGTAAGGATTGAACAAC (SEQ ID NO:21) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nAATGTTGTAGATACCTTTTTGTAAGGATTGAACAAC (SEQ ID NO:22) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2XX

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2XX and designated “Cas12L_51_82983331.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2XX. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2XX. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2XX. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2XX. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2XX, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 750 amino acids (aa) to 850 aa, e.g., from 750 aa to 779 aa, from 779 aa to 800 aa, or from 800 aa to 850 aa). In some cases, the Cas12L polypeptide has a length of 779 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2XX) includes the following nucleotide sequence: ATTGTTGTAATACTATTTTTGTAAAGTATAAACAAC (SEQ ID NO:23) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAATACTATTTTTGTAAAGTATAAACAAC (SEQ ID NO:24) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2YY

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2YY and designated “Cas12L_52_76767885.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2YY. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2YY. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2YY. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2YY. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2YY, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 550 amino acids (aa) to 700 aa, e.g., from 550 aa to 592 aa, from 592 aa to 625 aa, from 625 aa to 650 aa, or from 650 aa to 700 aa). In some cases, the Cas12L polypeptide has a length of 592 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2YY) includes the following nucleotide sequence: AATGTTGTAGATGCCTTTTTATAAGGATTAAACAAC (SEQ ID NO:25) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nAATGTTGTAGATGCCTTTTTATAAGGATTAAACAAC (SEQ ID NO:26) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2ZZ

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2ZZ and designated “Cas12L_53_77216451.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2ZZ. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2ZZ. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2ZZ. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2ZZ. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2ZZ, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2ZZ) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2AAA

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 29 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2AAA and designated “Cas12L_54_77468912_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 29 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2AAA. In some cases, a Cas12L protein includes a contiguous stretch of about 29 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2AAA. In some cases, a Cas12L protein includes a contiguous stretch of about 29 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2AAA. In some cases, a Cas12L protein includes a contiguous stretch of about 29 amino acids having the Cas12L amino acid sequence depicted in FIG. 2AAA. In some cases, a Cas12L protein includes a contiguous stretch of about 29 amino acids having the Cas12L protein sequence depicted in FIG. 2AAA, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2AAA) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2BBB

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2BBB and designated “Cas12L_55_77738117.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2BBB. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2BBB. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2BBB. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2BBB. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2BBB, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2BBB) includes the following nucleotide sequence: ATTGTTGTAATACACTTTTTATAAGGTATGAACAAC (SEQ ID NO:27) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAATACACTTTTTATAAGGTATGAACAAC (SEQ ID NO:28) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2CCC

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2CCC and designated “Cas12L_56_65286425.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2CCC. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2CCC. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2CCC. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2CCC. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2CCC, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 650 amino acids (aa) to 750 aa, e.g., from 650 aa to 692 aa, from 692 aa to 725 aa, or from 725 aa to 750 aa). In some cases, the Cas12L polypeptide has a length of 692 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2CCC) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2DDD

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 441 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2DDD and designated “Cas12L_57_65567118_partial.” For example, in some cases, a Cas12L protein) includes a contiguous stretch of about 441 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2DDD. In some cases, a Cas12L protein) includes a contiguous stretch of about 441 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2DDD. In some cases, a Cas12L protein) includes a contiguous stretch of about 441 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2DDD. In some cases, a Cas12L protein) includes a contiguous stretch of about 441 amino acids having the Cas12L amino acid sequence depicted in FIG. 2DDD. In some cases, a Cas12L protein) includes a contiguous stretch of about 441 amino acids having the Cas12L protein sequence depicted in FIG. 2DDD, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2DDD) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2EEE

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes a contiguous stretch of about 397 amino acids having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2EEE and designated “Cas12L_58_66287853_partial.” For example, in some cases, a Cas12L protein includes a contiguous stretch of about 397 amino acids having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2EEE. In some cases, a Cas12L protein includes a contiguous stretch of about 397 amino acids having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2EEE. In some cases, a Cas12L protein includes a contiguous stretch of about 397 amino acids having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2EEE. In some cases, a Cas12L protein includes a contiguous stretch of about 397 amino acids having the Cas12L amino acid sequence depicted in FIG. 2EEE. In some cases, a Cas12L protein includes a contiguous stretch of about 397 amino acids having the Cas12L protein sequence depicted in FIG. 2EEE, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 750 aa, from 750 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of from 725 amino acids to 775 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2EEE) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

FIG. 2FFF

In some cases, a Cas12L protein (of the subject compositions and/or methods) includes an amino acid sequence having 20% or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2FFF and designated “Cas12L_39_73877227.” For example, in some cases, a Cas12L protein includes an amino acid sequence having 50% or more sequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2FFF. In some cases, a Cas12L protein includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2FFF. In some cases, a Cas12L protein includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity) with the Cas12L amino acid sequence depicted in FIG. 2FFF. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L amino acid sequence depicted in FIG. 2FFF. In some cases, a Cas12L protein includes an amino acid sequence having the Cas12L protein sequence depicted in FIG. 2FFF, with the exception that the sequence includes an amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions) that reduces the naturally occurring catalytic activity of the protein. In some cases, the Cas12L polypeptide has a length of from 700 amino acids (aa) to 800 aa, e.g., from 700 aa to 725 aa, from 725 aa to 746 aa, from 746 aa to 775 aa, or from 775 aa to 800 aa). In some cases, the Cas12L polypeptide has a length of 746 amino acids. In some cases, a guide RNA that binds a Cas12L polypeptide (e.g., a Cas12L polypeptide comprising an amino acid sequence having 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%, amino acid sequence identity to the Cas12L amino acid sequence depicted in FIG. 2FFF) includes the following nucleotide sequence: ATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:1) or the reverse complement of same. In some cases, the guide RNA comprises the nucleotide sequence (N)nATTGTTGTAACTCTTATTTTGTATGGAGTAAACAAC (SEQ ID NO:2) or the reverse complement of same, where N is any nucleotide and n is an integer from 15 to 30, e.g., from 15 to 20, from 17 to 25, from 17 to 22, from 18 to 22, from 18 to 20, from 20 to 25, or from 25 to 30).

Cas12L Variants

A variant Cas12L protein has an amino acid sequence that is different by at least one amino acid (e.g., has a deletion, insertion, substitution, fusion) when compared to the amino acid sequence of the corresponding wild type Cas12L protein, e.g., when compared to the Cas12L amino acid sequence depicted in any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF. In some cases, a Cas12L variant comprises from 1 amino acid substitution to 10 amino acid substitutions compared to the Cas12L amino acid sequence depicted in any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF. In some cases, a Cas12L variant comprises from 1 amino acid substitution to 10 amino acid substitutions in the RuvC domain, compared to the Cas12L amino acid sequence depicted in any one of FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIG. 2AAA-2FFF.

Variants—Catalytic Activity

In some cases, the Cas12L protein is a variant Cas12L protein, e.g., mutated relative to the naturally occurring catalytically active sequence, and exhibits reduced cleavage activity (e.g., exhibits 90%, or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less cleavage activity) when compared to the corresponding naturally occurring sequence. In some cases, such a variant Cas12L protein is a catalytically ‘dead’ protein (has substantially no cleavage activity) and can be referred to as a ‘dCas12L.’ In some cases, the variant Cas12L protein is a nickase (cleaves only one strand of a double stranded target nucleic acid, e.g., a double stranded target DNA). As described in more detail herein, in some cases, a Cas12L protein (in some case a Cas12L protein with wild type cleavage activity and in some cases a variant Cas12L with reduced cleavage activity, e.g., a dCas12L or a nickase Cas12L) is fused (conjugated) to a heterologous polypeptide that has an activity of interest (e.g., a catalytic activity of interest) to form a fusion protein (a fusion Cas12L protein).

In some cases, a variant Cas12L polypeptide comprises a substitution of one, two, or three amino acids of the active site residues indicated in FIG. 3A-3B, where the variant Cas12L polypeptide exhibits reduced catalytic activity compared to a control Cas12L polypeptide that does not include the one, two, or three substitutions.

Variants—Fusion Cas12L Polypeptides

As noted above, in some cases, a Cas12L protein (in some cases a Cas12L protein with wild type cleavage activity and in some cases a variant Cas12L with reduced cleavage activity, e.g., a dCas12L or a nickase Cas12L) is fused (conjugated) to a heterologous polypeptide that has an activity of interest (e.g., a catalytic activity of interest) to form a fusion protein. A heterologous polypeptide to which a Cas12L protein can be fused is referred to herein as a ‘fusion partner.’

In some cases, the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA. For example, in some cases the fusion partner is a protein (or a domain from a protein) that inhibits transcription (e.g., a transcriptional repressor, a protein that functions via recruitment of transcription inhibitor proteins, modification of target DNA such as methylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like). In some cases the fusion partner is a protein (or a domain from a protein) that increases transcription (e.g., a transcription activator, a protein that acts via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like).

In some cases, the fusion partner (heterologous polypeptide) is a reverse transcriptase. In some cases, the fusion partner is a base editor. In some cases, the fusion partner (heterologous polypeptide) is a deaminase.

In some cases, a fusion Cas12L protein includes a heterologous polypeptide that has enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity).

In some cases, a fusion Cas12L protein includes a heterologous polypeptide that has enzymatic activity that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity).

Examples of proteins (or fragments thereof) that can be used in increase transcription include but are not limited to: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and/or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, and the like; histone lysine demethylases such as JHDM2a/b, UTX, JMJD3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, SRC1, ACTR, P160, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like.

Examples of proteins (or fragments thereof) that can be used in decrease transcription include but are not limited to: transcriptional repressors such as the Kruppel associated box (KRAB or SKD); KOX1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as Pr-SET7/8, SUV4-20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like; DNA methylases such as HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.

In some cases, the fusion partner has enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activity that can be provided by the fusion partner include but are not limited to: nuclease activity such as that provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like), DNA repair activity, DNA damage activity, deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme such as rat APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and/or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase; and the like), transposase activity, recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity).

In some cases, the fusion partner has enzymatic activity that modifies a protein associated with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein, and the like). Examples of enzymatic activity (that modifies a protein associated with a target nucleic acid) that can be provided by the fusion partner include but are not limited to: methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET/SETDB1, and the like, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7/8, SUV4-20H1, EZH2, RIZ1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a/b, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, UTX, JMJD3, and the like), acetyltransferase activity such as that provided by a histone acetylase transferase (e.g., catalytic core/fragement of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, HBO1/MYST2, HMOF/MYST1, SRC1, ACTR, P160, CLOCK, and the like), deacetylase activity such as that provided by a histone deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like), kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity.

Additional examples of a suitable fusion partners are dihydrofolate reductase (DHFR) destabilization domain (e.g., to generate a chemically controllable fusion Cas12L protein), and a chloroplast transit peptide. Suitable chloroplast transit peptides include, but are not limited to:

(SEQ ID NO: 29)

MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSITS

NGGRVKCMQVWPPIGKKKFETLSYLPPLTRDSRA;

(SEQ ID NO: 30)

MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSITS

NGGRVKS;

(SEQ ID NO: 31)

MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNG

GRVNCMQVWPPIEKKKFETLSYLPDLTDSGGRVNC;

(SEQ ID NO: 32)

MAQVSRICNGVQNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWG

LKKSGMTLIGSELRPLKVMSSVSTAC;

(SEQ ID NO: 33)

MAQVSRICNGVWNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWG

LKKSGMTLIGSELRPLKVMSSVSTAC;

(SEQ ID NO: 34)

MAQINNMAQGIQTLNPNSNFHKPQVPKSSSFLVFGSKKLKNSANSMLVLK

KDSIFMQLFCSFRISASVATAC;

(SEQ ID NO: 35)

MAALVTSQLATSGTVLSVTDRFRRPGFQGLRPRNPADAALGMRTVGASAA

PKQSRKPHRFDRRCLSMVV;

(SEQ ID NO: 36)

MAALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAGGDATSLSV

TTSARATPKQQRSVQRGSRRFPSVVVC;

(SEQ ID NO: 37)

MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIAS

NGGRVQC;

(SEQ ID NO: 38)

MESLAATSVFAPSRVAVPAARALVRAGTVVPTRRTSSTSGTSGVKCSAAV

TPQASPVISRSAAAA;

and

(SEQ ID NO: 39)

MGAAATSMQSLKFSNRLVPPSRRLSPVPNNVTCNNLPKSAAPVRTVKCCA

SSWNSTINGAAATTNGASAASS.

In some case, a Cas12L fusion polypeptide of the present disclosure comprises: a) a Cas12L polypeptide of the present disclosure; and b) a chloroplast transit peptide. Thus, for example, a Cas12L polypeptide/guide RNA complex can be targeted to the chloroplast. In some cases, this targeting may be achieved by the presence of an N-terminal extension, called a chloroplast transit peptide (CTP) or plastid transit peptide. Chromosomal transgenes from bacterial sources must have a sequence encoding a CTP sequence fused to a sequence encoding an expressed polypeptide if the expressed polypeptide is to be compartmentalized in the plant plastid (e.g. chloroplast). Accordingly, localization of an exogenous polypeptide to a chloroplast is often 1 accomplished by means of operably linking a polynucleotide sequence encoding a CTP sequence to the 5′ region of a polynucleotide encoding the exogenous polypeptide. The CTP is removed in a processing step during translocation into the plastid. Processing efficiency may, however, be affected by the amino acid sequence of the CTP and nearby sequences at the amino terminus (NH₂terminus) of the peptide. Other options for targeting to the chloroplast which have been described are the maize cab-m7 signal sequence (U.S. Pat. No. 7,022,896, WO 97/41228) a pea glutathione reductase signal sequence (WO 97/41228) and the CTP described in US2009029861.

In some cases, a Cas12L fusion polypeptide of the present disclosure can comprise: a) a Cas12L polypeptide of the present disclosure; and b) an endosomal escape peptide. In some cases, an endosomal escape polypeptide comprises the amino acid sequence GLFXALLXLLXSLWXLLLXA (SEQ ID NO:72), wherein each X is independently selected from lysine, histidine, and arginine. In some cases, an endosomal escape polypeptide comprises the amino acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO:73).

For examples of some of the above fusion partners (and more) used in the context of fusions with Cas9, Zinc Finger, and/or TALE proteins (for site specific target nucleic modification, modulation of transcription, and/or target protein modification, e.g., histone modification), see, e.g.: Nomura et al, J Am Chem Soc. 2007 Jul. 18;129(28):8676-7; Rivenbark et al., Epigenetics. 2012 April; 7(4):350-60; Nucleic Acids Res. 2016 Jul. 8;44(12):5615-28; Gilbert et al., Cell. 2013 Jul. 18;154(2):442-51; Kearns et al., Nat Methods. 2015 May; 12(5):401-3; Mendenhall et al., Nat Biotechnol. 2013 December; 31(12):1133-6; Hilton et al., Nat Biotechnol. 2015 May; 33(5):510-7; Gordley et al., Proc Natl Acad Sci USA. 2009 Mar. 31;106(13):5053-8; Akopian et al., Proc Natl Acad Sci USA. 2003 Jul. 22;100(15):8688-91; Tan et., al., J Virol. 2006 Feb;80(4):1939-48; Tan et al., Proc Natl Acad Sci USA. 2003 Oct. 14;100(21):11997-2002; Papworth et al., Proc Natl Acad Sci USA. 2003 Feb. 18;100(4):1621-6; Sanjana et al., Nat Protoc. 2012 Jan. 5;7(1):171-92; Beerli et al., Proc Natl Acad Sci USA. 1998 Dec 8;95(25):14628-33; Snowden et al., Curr Biol. 2002 Dec. 23;12(24):2159-66; Xu et.al., Xu et al., Cell Discov. 2016 May 3; 2:16009; Komor et al., Nature. 2016 Apr. 20;533(7603):420-4; Chaikind et al., Nucleic Acids Res. 2016 Aug. 11; Choudhury at. al., Oncotarget. 2016 Jun. 23; Du et al., Cold Spring Harb Protoc. 2016 Jan. 4; Pham et al., Methods Mol Biol. 2016; 1358:43-57; Balboa et al., Stem Cell Reports. 2015 Sep. 8;5(3):448-59; Hara et al., Sci Rep. 2015 Jun. 9; 5:11221; Piatek et al., Plant Biotechnol J. 2015 May; 13(4):578-89; Hu et al., Nucleic Acids Res. 2014 April; 42(7):4375-90; Cheng et al., Cell Res. 2013 October;23(10):1163-71; and Maeder et al., Nat Methods. 2013 October;10(10):977-9.

Additional suitable heterologous polypeptides include, but are not limited to, a polypeptide that directly and/or indirectly provides for increased transcription and/or translation of a target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription and/or translation regulator, a translation-regulating protein, etc.). Non-limiting examples of heterologous polypeptides to accomplish increased or decreased transcription include transcription activator and transcription repressor domains. In some such cases, a fusion Cas12L polypeptide is targeted by the guide nucleic acid (guide RNA) to a specific location (i.e., sequence) in the target nucleic acid and exerts locus-specific regulation such as blocking RNA polymerase binding to a promoter (which selectively inhibits transcription activator function), and/or modifying the local chromatin status (e.g., when a fusion sequence is used that modifies the target nucleic acid or modifies a polypeptide associated with the target nucleic acid). In some cases, the changes are transient (e.g., transcription repression or activation). In some cases, the changes are inheritable (e.g., when epigenetic modifications are made to the target nucleic acid or to proteins associated with the target nucleic acid, e.g., nucleosomal histones).

Non-limiting examples of heterologous polypeptides for use when targeting ssRNA target nucleic acids include (but are not limited to): splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and/or release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and/or C to U editing enzymes); helicases; RNA-binding proteins; and the like. It is understood that a heterologous polypeptide can include the entire protein or in some cases can include a fragment of the protein (e.g., a functional domain).

The heterologous polypeptide of a subject fusion Cas12L polypeptide can be any domain capable of interacting with ssRNA (which, for the purposes of this disclosure, includes intramolecular and/or intermolecular secondary structures, e.g., double-stranded RNA duplexes such as hairpins, stem-loops, etc.), whether transiently or irreversibly, directly or indirectly, including but not limited to an effector domain selected from the group comprising; Endonucleases (for example RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus) domains from proteins such as SMG5 and SMG6); proteins and protein domains responsible for stimulating RNA cleavage (for example CPSF, CstF, CFIm and CFIIm); Exonucleases (for example XRN-1 or Exonuclease T); Deadenylases (for example HNT3); proteins and protein domains responsible for nonsense mediated RNA decay (for example UPF1, UPF2, UPF3, UPF3b, RNP Si, Y14, DEK, REF2, and SRm160); proteins and protein domains responsible for stabilizing RNA (for example PABP); proteins and protein domains responsible for repressing translation (for example Ago2 and Ago4); proteins and protein domains responsible for stimulating translation (for example Staufen); proteins and protein domains responsible for (e.g., capable of) modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains responsible for polyadenylation of RNA (for example PAP1, GLD-2, and Star-PAP); proteins and protein domains responsible for polyuridinylation of RNA (for example CI D1 and terminal uridylate transferase); proteins and protein domains responsible for RNA localization (for example from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins and protein domains responsible for nuclear retention of RNA (for example Rrp6); proteins and protein domains responsible for nuclear export of RNA (for example TAP, NXF1, THO, TREX, REF, and Aly); proteins and protein domains responsible for repression of RNA splicing (for example PTB, Sam68, and hnRNP A1); proteins and protein domains responsible for stimulation of RNA splicing (for example Serine/Arginine-rich (SR) domains); proteins and protein domains responsible for reducing the efficiency of transcription (for example FUS (TLS)); and proteins and protein domains responsible for stimulating transcription (for example CDK7 and HIV Tat). Alternatively, the effector domain may be selected from the group comprising Endonucleases; proteins and protein domains capable of stimulating RNA cleavage; Exonucleases; Deadenylases; proteins and protein domains having nonsense mediated RNA decay activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of repressing translation; proteins and protein domains capable of stimulating translation; proteins and protein domains capable of modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridinylation of RNA; proteins and protein domains having RNA localization activity; proteins and protein domains capable of nuclear retention of RNA; proteins and protein domains having RNA nuclear export activity; proteins and protein domains capable of repression of RNA splicing; proteins and protein domains capable of stimulation of RNA splicing; proteins and protein domains capable of reducing the efficiency of transcription; and proteins and protein domains capable of stimulating transcription. Another suitable heterologous polypeptide is a PUF RNA-binding domain, which is described in more detail in WO2012068627, which is hereby incorporated by reference in its entirety.

Some RNA splicing factors that can be used (in whole or as fragments thereof) as heterologous polypeptides for a fusion Cas12L polypeptide have modular organization, with separate sequence-specific RNA binding modules and splicing effector domains. For example, members of the Serine/Arginine-rich (SR) protein family contain N-terminal RNA recognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and C-terminal RS domains that promote exon inclusion. As another example, the hnRNP protein hnRNP A1 binds to exonic splicing silencers (ESSs) through its RRM domains and inhibits exon inclusion through a C-terminal Glycine-rich domain. Some splicing factors can regulate alternative use of splice site (ss) by binding to regulatory sequences between the two alternative sites. For example, ASF/SF2 can recognize ESEs and promote the use of intron proximal sites, whereas hnRNP A1 can bind to ESSs and shift splicing towards the use of intron distal sites. One application for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly disease associated genes. For example, Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5′ splice sites to encode proteins of opposite functions. The long splicing isoform Bcl-xL is a potent apoptosis inhibitor expressed in long-lived postmitotic cells and is up-regulated in many cancer cells, protecting cells against apoptotic signals. The short isoform Bcl-xS is a pro-apoptotic isoform and expressed at high levels in cells with a high turnover rate (e.g., developing lymphocytes). The ratio of the two Bcl-x splicing isoforms is regulated by multiple cω-elements that are located in either the core exon region or the exon extension region (i.e., between the two alternative 5′ splice sites). For more examples, see WO2010075303, which is hereby incorporated by reference in its entirety.

Further suitable fusion partners include, but are not limited to, proteins (or fragments thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that provide periphery recruitment (e.g., Lamin A, Lamin B, etc.), protein docking elements (e.g., FKBP/FRB, Pill/Abyl, etc.).

Nucleases

In some cases, a subject fusion Cas12L polypeptide comprises: i) a Cas12L polypeptide of the present disclosure; and ii) a heterologous polypeptide (a “fusion partner”), where the heterologous polypeptide is a nuclease. Suitable nucleases include, but are not limited to, a homing nuclease polypeptide; a FokI polypeptide; a transcription activator-like effector nuclease (TALEN) polypeptide; a MegaTAL polypeptide; a meganuclease polypeptide; a zinc finger nuclease (ZFN); an ARCUS nuclease; and the like. The meganuclease can be engineered from an LADLIDADG homing endonuclease (LHE). A megaTAL polypeptide can comprise a TALE DNA binding domain and an engineered meganuclease. See, e.g., WO 2004/067736 (homing endonuclease); Urnov et al. (2005) Nature 435:646 (ZFN); Mussolino et al. (2011) Nucle. Acids Res. 39:9283 (TALE nuclease); Boissel et al. (2013) Nucl. Acids Res. 42:2591 (MegaTAL).

Reverse Transcriptases

In some cases, a subject fusion Cas12L polypeptide comprises: i) a Cas12L polypeptide of the present disclosure; and ii) a heterologous polypeptide (a “fusion partner”), where the heterologous polypeptide is a reverse transcriptase polypeptide. In some cases, the Cas12L polypeptide is catalytically inactive. Suitable reverse transcriptases include, e.g., a murine leukemia virus reverse transcriptase; a Rous sarcoma virus reverse transcriptase; a human immunodeficiency virus type I reverse transcriptase; a Moloney murine leukemia virus reverse transcriptase; and the like.

Base Editors

In some cases, a Cas12L fusion polypeptide of the present disclosure comprises: i) a Cas12L polypeptide of the present disclosure; and ii) a heterologous polypeptide (a “fusion partner”), where the heterologous polypeptide is a base editor. Suitable base editors include, e.g., an adenosine deaminase; a cytidine deaminase (e.g., an activation-induced cytidine deaminase (AID)); APOBEC3G; and the like); and the like.

A suitable adenosine deaminase is any enzyme that is capable of deaminating adenosine in DNA. In some cases, the deaminase is a TadA deaminase.

In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

(SEQ ID NO: 74)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPI

GRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSR

IGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSD

FFRMRRQEIKAQKKAQSSTD

In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino

(SEQ ID NO: 75)

MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNN

RVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPC

VMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGI

LADECAALLSDFFRMRRQEIKAQKKAQSSTD.

In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Staphylococcus aureus TadA amino acid sequence:

(SEQ ID NO: 76)

MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRE

TLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSR

IPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTT

FFKNLRANKKSTN:

In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Bacillus subtilis TadA amino acid sequence:

(SEQ ID NO: 77)

MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRS

IAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVF

GAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRK

KKKAARKNLSE

In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Salmonella typhimurium TadA:

(SEQ ID NO: 78)

MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHR

VIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVM

CAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRD

ECATLLSDFFRMRRQEIKALKKADRAEGAGPAV

In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Shewanella putrefaciens TadA amino acid sequence:

(SEQ ID NO: 79)

MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTA

HAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGA

RDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEK

KALKLAQRAQQGIE

In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Haemophilus influenzae F3031 TadA amino acid sequence:

(SEQ ID NO: 80)

MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWN

LSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILH

SRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLS

TFFQKRREEKKIEKALLKSLSDK

In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Caulobacter crescentus TadA amino acid sequence:

(SEQ ID NO: 81)

MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAG

NGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAI

SHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESAD

LLRGFFRARRKAKI

In some cases, a suitable adenosine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following Geobacter sulfurreducens TadA amino acid sequence:

(SEQ ID NO: 82)

MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGH

NLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAI

ILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGT

MLSDFFRDLRRRKKAKATPALFIDERKVPPEP

Cytidine deaminases suitable for inclusion in a CRISPR/Cas effector polypeptide fusion polypeptide include any enzyme that is capable of deaminating cytidine in DNA.

In some cases, the cytidine deaminase is a deaminase from the apolipoprotein B mRNA-editing complex (APOBEC) family of deaminases. In some cases, the APOBEC family deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In some cases, the cytidine deaminase is an activation induced deaminase (AID).

In some cases, a suitable cytidine deaminase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

(SEQ ID NO: 83)

MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGY

LRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVAD

FLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDY

FYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDA

FRTLGL

In some cases, a suitable cytidine deaminase is an AID and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSAT SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKENHERTFK AWEGLHENSV RLSRQLRRIL LPLYEVDDLR DAFRTLGL (SEQ ID NO:84).

In some cases, a suitable cytidine deaminase is an AID and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDSLLMNRRK FLYQFKNVRW AKGRRETYLC YVVKRRDSAT SFSLDFGYLR NKNGCHVELL FLRYISDWDL DPGRCYRVTW FTSWSPCYDC ARHVADFLRG NPNLSLRIFT ARLYFCEDRK AEPEGLRRLH RAGVQIAIMT FKDYFYCWNT FVENHERTFK AWEGLHENSV RLSRQLRRIL LPLYEVDDLR DAFRTLGL (SEQ ID NO:85).

Transcription Factors

In some cases, a Cas12L fusion polypeptide of the present disclosure comprises: i) a Cas12L polypeptide of the present disclosure; and ii) a heterologous polypeptide (a “fusion partner”), where the heterologous polypeptide is a transcription factor. A transcription factor can include: i) a DNA binding domain; and ii) a transcription activator. A transcription factor can include: i) a DNA binding domain; and ii) a transcription repressor. Suitable transcription factors include polypeptides that include a transcription activator or a transcription repressor domain (e.g., the Kruppel associated box (KRAB or SKD); the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), etc.); zinc-finger-based artificial transcription factors (see, e.g., Sera (2009) Adv. Drug Deliv. 61:513); TALE-based artificial transcription factors (see, e.g., Liu et al. (2013) Nat. Rev. Genetics 14:781); and the like. In some cases, the transcription factor comprises a VP64 polypeptide (transcriptional activation). In some cases, the transcription factor comprises a Kruppel-associated box (KRAB) polypeptide (transcriptional repression). In some cases, the transcription factor comprises a Mad mSIN3 interaction domain (SID) polypeptide (transcriptional repression). In some cases, the transcription factor comprises an ERF repressor domain (ERD) polypeptide (transcriptional repression). For example, in some cases, the transcription factor is a transcriptional activator, where the transcriptional activator is GAL4-VP16.

Recombinases

In some cases, a Cas12L fusion polypeptide of the present disclosure comprises: i) a Cas12L polypeptide of the present disclosure; and ii) a heterologous polypeptide (a “fusion partner”), where the heterologous polypeptide is a recombinase. Suitable recombinases include, e.g., a Cre recombinase; a Hin recombinase; a Tre recombinase; a FLP recombinase; and the like.

Examples of various additional suitable heterologous polypeptide (or fragments thereof) for a subject fusion Cas12L polypeptide include, but are not limited to, those described in the following applications (which publications are related to other CRISPR endonucleases such as Cas9, but the described fusion partners can also be used with Cas12L instead): PCT patent applications: WO2010075303, WO2012068627, and WO2013155555, and can be found, for example, in U.S. patents and patent applications: U.S. Pat. Nos. 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919; 20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; all of which are hereby incorporated by reference in their entirety.

In some cases, a heterologous polypeptide (a fusion partner) provides for subcellular localization, i.e., the heterologous polypeptide contains a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence to keep the fusion protein out of the nucleus, e.g., a nuclear export sequence (NES), a sequence to keep the fusion protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an ER retention signal, and the like). In some embodiments, a Cas12L fusion polypeptide does not include an NLS so that the protein is not targeted to the nucleus (which can be advantageous, e.g., when the target nucleic acid is an RNA that is present in the cytosol). In some embodiments, the heterologous polypeptide can provide a tag (i.e., the heterologous polypeptide is a detectable label) for ease of tracking and/or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and the like; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).

In some cases, a Cas12L protein (e.g., a wild type Cas12L protein, a variant Cas12L protein, a fusion Cas12L protein, a dCas12L protein, and the like) includes (is fused to) a nuclear localization signal (NLS) (e.g., in some cases 2 or more, 3 or more, 4 or more, or 5 or more NLSs). Thus, in some cases, a Cas12L polypeptide includes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and/or the C-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the C-terminus. In some cases, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some cases, an NLS is positioned at the N-terminus and an NLS is positioned at the C-terminus.

In some cases, a Cas12L protein (e.g., a wild type Cas12L protein, a variant Cas12L protein, a fusion Cas12L protein, a dCas12L protein, and the like) includes (is fused to) between 1 and 10 NLSs (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, or 2-5 NLSs). In some cases, a Cas12L protein (e.g., a wild type Cas12L protein, a variant Cas12L protein, a fusion Cas12L protein, a dCas12L protein, and the like) includes (is fused to) between 2 and 5 NLSs (e.g., 2-4, or 2-3 NLSs).

Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 86); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO:87)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO:88) or RQRRNELKRSP (SEQ ID NO:89); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:90); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:91) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 92) and PPKKARED (SEQ ID NO:93) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO:94) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO:95) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 96) and PKQKKRK (SEQ ID NO:97) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO:98) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO:99) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:100) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO:101) of the steroid hormone receptors (human) glucocorticoid. In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of the Cas12L protein in a detectable amount in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the Cas12L protein such that location within a cell may be visualized. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly.

In some cases, a Cas12L fusion polypeptide includes a “Protein Transduction Domain” or PTD (also known as a CPP—cell penetrating peptide), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from a small polar molecule to a large macromolecule and/or a nanoparticle, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In some embodiments, a PTD is covalently linked to the amino terminus a polypeptide (e.g., linked to a wild type Cas12L polypeptide to generate a fusion protein, or linked to a variant Cas12L protein such as a dCas12L, nickase Cas12L, or fusion Cas12L protein, to generate a fusion protein). In some embodiments, a PTD is covalently linked to the carboxyl terminus of a polypeptide (e.g., linked to a wild type Cas12L to generate a fusion protein, or linked to a variant Cas12L protein such as a dCas12L, nickase Cas12L, or fusion Cas12L protein to generate a fusion protein). In some cases, the PTD is inserted internally in the Cas12L fusion polypeptide (i.e., is not at the N- or C-terminus of the Cas12L fusion polypeptide) at a suitable insertion site. In some cases, a subject Cas12L fusion polypeptide includes (is conjugated to, is fused to) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some cases, a PTD includes a nuclear localization signal (NLS) (e.g., in some cases 2 or more, 3 or more, 4 or more, or 5 or more NLSs). Thus, in some cases, a Cas12L fusion polypeptide includes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). In some embodiments, a PTD is covalently linked to a nucleic acid (e.g., a Cas12L guide nucleic acid, a polynucleotide encoding a Cas12L guide nucleic acid, a polynucleotide encoding a Cas12L fusion polypeptide, a donor polynucleotide, etc.). Examples of PTDs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; SEQ ID NO:40); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:41); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:42); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:43); and RQIKIWFQNRRMKWKK (SEQ ID NO:44). Exemplary PTDs include but are not limited to, YGRKKRRQRRR (SEQ ID NO:40), RKKRRQRRR (SEQ ID NO:45); an arginine homopolymer of from 3 arginine residues to 50 arginine residues; Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO:40); RKKRRQRR (SEQ ID NO:46); YARAAARQARA (SEQ ID NO:47); THRLPRRRRRR (SEQ ID NO:48); and GGRRARRRRRR (SEQ ID NO:49). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”) connected via a cleavable linker to a matching polyanion (e.g., Glu9 or “E9”), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus “activating” the ACPP to traverse the membrane.

Linkers (e.g., for Fusion Partners)

In some embodiments, a subject Cas12L protein can fused to a fusion partner via a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use.

Examples of linker polypeptides include glycine polymers (G)_n, glycine-serine polymers (including, for example, (GS)_n, GSGGS_n(SEQ ID NO: 50), GGSGGS_n(SEQ ID NO:51), and GGGS_n(SEQ ID NO: 52), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers. Exemplary linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:53), GGSGG (SEQ ID NO:54), GSGSG (SEQ ID NO:55), GSGGG (SEQ ID NO:56), GGGSG (SEQ ID NO:57), GSSSG (SEQ ID NO: 58), and the like. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any desired element can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.

Detectable Labels

In some cases, a Cas12L polypeptide of the present disclosure comprises a detectable label. Suitable detectable labels and/or moieties that can provide a detectable signal can include, but are not limited to, an enzyme, a radioisotope, a member of a specific binding pair; a fluorophore; a fluorescent protein; a quantum dot; and the like.

Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitable for use.

Suitable enzymes include, but are not limited to, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, glucose oxidase (GO), and the like.

Protospacer Adjacent Motif (PAM)

A Cas12L protein binds to target DNA at a target sequence defined by the region of complementarity between the DNA-targeting RNA and the target DNA. As is the case for many CRISPR endonucleases, site-specific binding (and/or cleavage) of a double stranded target DNA occurs at locations determined by both (i) base-pairing complementarity between the guide RNA and the target DNA; and (ii) a short motif [referred to as the protospacer adjacent motif (PAM)] in the target DNA.

In some cases, the PAM for a Cas12L protein is immediately 5′ of the target sequence of the non-complementary strand of the target DNA (the complementary strand: (i) hybridizes to the guide sequence of the guide RNA, while the non-complementary strand does not directly hybridize with the guide RNA; and (ii) is the reverse complement of the non-complementary strand).

In some cases, different Cas12L proteins (i.e., Cas12L proteins from various species) may be advantageous to use in the various provided methods in order to capitalize on various enzymatic characteristics of the different Cas12L proteins (e.g., for different PAM sequence preferences; for increased or decreased enzymatic activity; for an increased or decreased level of cellular toxicity; to change the balance between NHEJ, homology-directed repair, single strand breaks, double strand breaks, etc.; to take advantage of a short total sequence; and the like). Cas12L proteins from different species may require different PAM sequences in the target DNA. Various methods (including in silico and/or wet lab methods) for identification of the appropriate PAM sequence are known in the art and are routine, and any convenient method can be used.

As shown in Example 1, a Cal12L polypeptide of the present disclosure can be reprogrammed (by complexing with a guide RNA) to cleave any sequence of a target nucleic acid (e.g., a target DNA) that is complementary to the targeting segment of the guide RNA, where the PAM is present on the 5′ end of the target (e.g., a T-rich PAM for Cas),); additional RNA components are not required for the formation of functional effectors in vivo. In some cases, a PAM sequence is a T-rich sequence (e.g., TTR, where R is a purine). In some cases, a PAM sequence is TTA. In some cases, a PAM sequence is TTG.

Cas12L Guide RNA

A nucleic acid that binds to a Cas12L protein, forming a ribonucleoprotein complex (RNP), and targets the complex to a specific location within a target nucleic acid (e.g., a target DNA) is referred to herein as a “Cas12L guide RNA” or simply as a “guide RNA.” It is to be understood that in some cases, a hybrid DNA/RNA can be made such that a Cas12L guide RNA includes DNA bases in addition to RNA bases, but the term “Cas12L guide RNA” is still used to encompass such a molecule herein.

A Cas12L guide RNA can be said to include two segments, a targeting segment and a protein-binding segment. The protein-binding segment is also referred to herein as the “constant region” of the guide RNA. The targeting segment of a Cas12L guide RNA includes a nucleotide sequence (a guide sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within a target nucleic acid (e.g., a target dsDNA, a target ssRNA, a target ssDNA, the complementary strand of a double stranded target DNA, etc.). The protein-binding segment (or “protein-binding sequence”) interacts with (binds to) a Cas12L polypeptide. The protein-binding segment of a subject Cas12L guide RNA can include two complementary stretches of nucleotides that hybridize to one another to form a double stranded RNA duplex (dsRNA duplex). Site-specific binding and/or cleavage of a target nucleic acid (e.g., genomic DNA, ds DNA, RNA, etc.) can occur at locations (e.g., target sequence of a target locus) determined by base-pairing complementarity between the Cas12L guide RNA (the guide sequence of the Cas12L guide RNA) and the target nucleic acid.

A Cas12L guide RNA and a Cas12L protein (e.g., a wild-type Cas12L protein; a variant Cas12L protein; a fusion Cas12L polypeptide; etc.) form a complex (e.g., bind via non-covalent interactions). The Cas12L guide RNA provides target specificity to the complex by including a targeting segment, which includes a guide sequence (a nucleotide sequence that is complementary to a sequence of a target nucleic acid). The Cas12L protein of the complex provides the site-specific activity (e.g., cleavage activity provided by the Cas12L protein and/or an activity provided by the fusion partner in the case of a fusion Cas12L protein). In other words, the Cas12L protein is guided to a target nucleic acid sequence (e.g. a target sequence) by virtue of its association with the Cas12L guide RNA.

The “guide sequence” also referred to as the “targeting sequence” of a Cas12L guide RNA can be modified so that the Cas12L guide RNA can target a Cas12L protein (e.g., a naturally occurring Cas12L protein, a fusion Cas12L polypeptide, and the like) to any desired sequence of any desired target nucleic acid, with the exception (e.g., as described herein) that the PAM sequence can be taken into account. Thus, for example, a Cas12L guide RNA can have a guide sequence with complementarity to (e.g., can hybridize to) a sequence in a nucleic acid in a eukaryotic cell, e.g., a viral nucleic acid, a eukaryotic nucleic acid (e.g., a eukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.), and the like.

Guide Sequence of a Cas12L Guide RNA

A subject Cas12L guide RNA includes a guide sequence (i.e., a targeting sequence), which is a nucleotide sequence that is complementary to a sequence (a target site) in a target nucleic acid. In other words, the guide sequence of a Cas12L guide RNA can interact with a target nucleic acid (e.g., double stranded DNA (dsDNA), single stranded DNA (ssDNA), single stranded RNA (ssRNA), or double stranded RNA (dsRNA)) in a sequence-specific manner via hybridization (i.e., base pairing). The guide sequence of a Cas12L guide RNA can be modified (e.g., by genetic engineering)/designed to hybridize to any desired target sequence (e.g., while taking the PAM into account, e.g., when targeting a dsDNA target) within a target nucleic acid (e.g., a eukaryotic target nucleic acid such as genomic DNA).

In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%). In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100%.

In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over the seven contiguous 3′-most nucleotides of the target site of the target nucleic acid.

In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or more) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or more) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or more) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over 17 or more (e.g., 18 or more, 19 or more, 20 or more, 21 or more, 22 or more) contiguous nucleotides.

In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 19 or more (e.g., 20 or more, 21 or more, 22 or more) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 19 or more (e.g., 20 or more, 21 or more, 22 or more) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 19 or more (e.g., 20 or more, 21 or more, 22 or more) contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over 19 or more (e.g., 20 or more, 21 or more, 22 or more) contiguous nucleotides.

In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 17-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 17-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 17-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over 17-25 contiguous nucleotides.

In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 60% or more (e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 19-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 19-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 100%) over 19-25 contiguous nucleotides. In some cases, the percent complementarity between the guide sequence and the target site of the target nucleic acid is 100% over 19-25 contiguous nucleotides.

In some cases, the guide sequence has a length in a range of from 17-30 nucleotides (nt) (e.g., from 17-25, 17-22, 17-20, 19-30, 19-25, 19-22, 19-20, 20-30, 20-25, or 20-22 nt). In some cases, the guide sequence has a length in a range of from 17-25 nucleotides (nt) (e.g., from 17-22, 17-20, 19-25, 19-22, 19-20, 20-25, or 20-22 nt). In some cases, the guide sequence has a length of 17 or more nt (e.g., 18 or more, 19 or more, 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some cases, the guide sequence has a length of 19 or more nt (e.g., 20 or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, etc.). In some cases, the guide sequence has a length of 17 nt. In some cases, the guide sequence has a length of 18 nt. In some cases, the guide sequence has a length of 19 nt. In some cases the guide sequence has a length of 20 nt. In some cases the guide sequence has a length of 21 nt. In some cases the guide sequence has a length of 22 nt. In some cases the guide sequence has a length of 23 nt.

In some cases, the guide sequence (also referred to as a “spacer sequence”) has a length of from 15 to 50 nucleotides (e.g., from 15 nucleotides (nt) to 20 nt, from 20 nt to 25 nt, from 25 nt to 30 nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt, or from 45 nt to 50 nt).

Protein-Binding Segment of a Cas12L Guide RNA

The protein-binding segment (the “constant region”) of a subject Cas12L guide RNA interacts with a Cas12L protein. The Cas12L guide RNA guides the bound Cas12L protein to a specific nucleotide sequence within target nucleic acid via the above-mentioned guide sequence. The protein-binding segment of a Cas12L guide RNA can include two stretches of nucleotides that are complementary to one another and hybridize to form a double stranded RNA duplex (dsRNA duplex). Thus, in some cases, the protein-binding segment includes a dsRNA duplex.

In some cases, the dsRNA duplex region includes a range of from 5-25 base pairs (bp) (e.g., from 5-22, 5-20, 5-18, 5-15, 5-12, 5-10, 5-8, 8-25, 8-22, 8-18, 8-15, 8-12, 12-25, 12-22, 12-18, 12-15, 13-25, 13-22, 13-18, 13-15, 14-25, 14-22, 14-18, 14-15, 15-25, 15-22, 15-18, 17-25, 17-22, or 17-18 bp, e.g., 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). In some cases, the dsRNA duplex region includes a range of from 6-15 base pairs (bp) (e.g., from 6-12, 6-10, or 6-8 bp, e.g., 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, etc.). In some cases, the duplex region includes 5 or more bp (e.g., 6 or more, 7 or more, or 8 or more bp). In some cases, the duplex region includes 6 or more bp (e.g., 7 or more, or 8 or more bp). In some cases, not all nucleotides of the duplex region are paired, and therefore the duplex forming region can include a bulge. The term “bulge” herein is used to mean a stretch of nucleotides (which can be one nucleotide) that do not contribute to a double stranded duplex, but which are surround 5′ and 3′ by nucleotides that do contribute, and as such a bulge is considered part of the duplex region. In some cases, the dsRNA includes 1 or more bulges (e.g., 2 or more, 3 or more, 4 or more bulges). In some cases, the dsRNA duplex includes 2 or more bulges (e.g., 3 or more, 4 or more bulges). In some cases, the dsRNA duplex includes 1-5 bulges (e.g., 1-4, 1-3, 2-5, 2-4, or 2-3 bulges).

Thus, in some cases, the stretches of nucleotides that hybridize to one another to form the dsRNA duplex have 70%-100% complementarity (e.g., 75%-100%, 80%-10%, 85%-100%, 90%-100%, 95%-100% complementarity) with one another. In some cases, the stretches of nucleotides that hybridize to one another to form the dsRNA duplex have 70%-100% complementarity (e.g., 75%-100%, 80%-10%, 85%-100%, 90%-100%, 95%-100% complementarity) with one another. In some cases, the stretches of nucleotides that hybridize to one another to form the dsRNA duplex have 85%-100% complementarity (e.g., 90%-100%, 95%-100% complementarity) with one another. In some cases, the stretches of nucleotides that hybridize to one another to form the dsRNA duplex have 70%-95% complementarity (e.g., 75%-95%, 80%-95%, 85%-95%, 90%-95% complementarity) with one another.

In other words, in some embodiments, the dsRNA duplex includes two stretches of nucleotides that have 70%-100% complementarity (e.g., 75%-100%, 80%-10%, 85%-100%, 90%-100%, 95%-100% complementarity) with one another. In some cases, the dsRNA duplex includes two stretches of nucleotides that have 85%-100% complementarity (e.g., 90%-100%, 95%-100% complementarity) with one another. In some cases, the dsRNA duplex includes two stretches of nucleotides that have 70%-95% complementarity (e.g., 75%-95%, 80%-95%, 85%-95%, 90%-95% complementarity) with one another.

The duplex region of a subject Cas12L guide RNA can include one or more (1, 2, 3, 4, 5, etc) mutations relative to a naturally occurring duplex region. For example, in some cases a base pair can be maintained while the nucleotides contributing to the base pair from each segment can be different. In some cases, the duplex region of a subject Cas12L guide RNA includes more paired bases, less paired bases, a smaller bulge, a larger bulge, fewer bulges, more bulges, or any convenient combination thereof, as compared to a naturally occurring duplex region (of a naturally occurring Cas12L guide RNA).

Examples of various Cas9 guide RNAs can be found in the art, and in some cases variations similar to those introduced into Cas9 guide RNAs can also be introduced into Cas12L guide RNAs of the present disclosure (e.g., mutations to the dsRNA duplex region, extension of the 5′ or 3′ end for added stability for to provide for interaction with another protein, and the like). For example, see Jinek et al., Science. 2012 Aug. 17;337(6096):816-21; Chylinski et al., RNA Biol. 2013 May; 10(5):726-37; Ma et al., Biomed Res Int. 2013; 2013:270805; Hou et al., Proc Natl Acad Sci USA. 2013 Sep. 24;110(39):15644-9; Jinek et al., Elife. 2013;2:e00471; Pattanayak et al., Nat Biotechnol. 2013 September;31(9):839-43; Qi et al, Cell. 2013 Feb 28;152(5):1173-83; Wang et al., Cell. 2013 May 9;153(4):910-8; Auer et al., Genome Res. 2013 Oct. 31; Chen et al., Nucleic Acids Res. 2013 Nov. 1;41(20):e19; Cheng et al., Cell Res. 2013 Oct;23(10):1163-71; Cho et al., Genetics. 2013 November; 195(3):1177-80; DiCarlo et al., Nucleic Acids Res. 2013 April; 41(7):4336-43; Dickinson et al., Nat Methods. 2013 October;10(10):1028-34; Ebina et al., Sci Rep. 2013; 3:2510; Fujii et. al, Nucleic Acids Res. 2013 Nov. 1;41(20):e187; Hu et al., Cell Res. 2013 November; 23(11):1322-5; Jiang et al., Nucleic Acids Res. 2013 Nov. 1;41(20):e188; Larson et al., Nat Protoc. 2013 November; 8(11):2180-96; Mali et. at., Nat Methods. 2013 Oct;10(10):957-63; Nakayama et al., Genesis. 2013 December; 51(12):835-43; Ran et al., Nat Protoc. 2013 November; 8(11):2281-308; Ran et al., Cell. 2013 Sep. 12;154(6):1380-9; Upadhyay et al., G3 (Bethesda). 2013 Dec. 9;3(12):2233-8; Walsh et al., Proc Natl Acad Sci USA. 2013 Sep. 24;110(39):15514-5; Xie et al., Mol Plant. 2013 Oct. 9; Yang et al., Cell. 2013 Sep. 12;154(6):1370-9; Briner et al., Mol Cell. 2014 Oct. 23;56(2):333-9; and U.S. patents and patent applications: U.S. Pat. Nos. 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919; 20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956;20140356958;20140356959;20140357523;20140357530;20140364333;and 20140377868; all of which are hereby incorporated by reference in their entirety.

Examples of constant regions suitable for inclusion in a Cas12L guide RNA are provided in FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF (e.g., where T is substituted with U). A Cas12L guide RNA can include a constant region having from 1 to 5 nucleotide substitutions compared to any one of the nucleotide sequences depicted in FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF.

The nucleotide sequences (with T substituted with U) can be combined with a spacer sequence (where the spacer sequence comprises a target nucleic acid-binding sequence (“guide sequence”)) of choice that is from 15 to 50 nucleotides (e.g., from 15 nucleotides (nt) to 20 nt, from 20 nt to 25 nt, from 25 nt to 30 nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt, or from 45 nt to 50 nt in length). In some cases, the spacer sequence is 35-38 nucleotides in length. For example, any one of the nucleotide sequences (with T substituted with U) depicted in FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF can be included in a guide RNA comprising (N)n-constant region, where N is any nucleotide and n is an integer from 15 to 50 (e.g., from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 35, from 35 to 38, from 35 to 40, from 40 to 45, or from 45 to 50).

As one example, the constant region of a Cas12L guide RNA can comprise the nucleotide sequence: AUUGUUGUAACUCUUAUUUUGUAUGGAGUAAACAAC (SEQ ID NO:59). As another example, the constant region of a Cas12L guide RNA can comprise the nucleotide sequence: AUUGUUGUAGACCUCUUUUUAUAAGGAUUGAACAAC (SEQ ID NO:60; see FIG. 2B). As another example, the constant region of a Cas12L guide RNA can comprise the nucleotide sequence: UAUUGUUGUAGAUACCUUUUUGUAAGGAUUAAACAAC (SEQ ID NO:61; see FIG. 2P). As another example, the constant region of a Cas12L guide RNA can comprise the nucleotide sequence: AAUGUUGUAGAUGCCUUUUUAUAAGGAUUAAACAACUUG (SEQ ID NO:62; see FIG. 2T). As another example, the constant region of a Cas12L guide RNA can comprise the nucleotide sequence: AUUGUUGAAAUAGUACUUUUAUAGUCUAUAUACAAC (SEQ ID NO:63; see FIG. 2BB). As another example, the constant region of a Cas12L guide RNA can comprise the nucleotide sequence: UAUUGUUGUAACUCUUAUUUUGUAUGGAGUAAACAAC (SEQ ID NO:64; see FIG. 2CC). As another example, the constant region of a Cas12L guide RNA can comprise the nucleotide sequence: AUUGUUGUAACUUUUAUUUUGUAUGGAGUAAACAAC (SEQ ID NO:65; see FIG. 2TT). As another example, the constant region of a Cas12L guide RNA can comprise the nucleotide sequence: AAUGUUGUAGAUACCUUUUUGUAAGGAUUGAACAAC (SEQ ID NO:66; see FIG. 2WW). As another example, the constant region of a Cas12L guide RNA can comprise the nucleotide sequence: AUUGUUGUAAUACUAUUUUUGUAAAGUAUAAACAAC (SEQ ID NO:67; see FIG. 2XX). As another example, the constant region of a Cas12L guide RNA can comprise the nucleotide sequence: AAUGUUGUAGAUGCCUUUUUAUAAGGAUUAAACAAC (SEQ ID NO:68; see FIG. 2YY). As another example, the constant region of a Cas12L guide RNA can comprise the nucleotide sequence: AUUGUUGUAAUACACUUUUUAUAAGGUAUGAACAAC (SEQ ID NO:69; see FIG. 2BBB). As another example, the constant region of a Cas12L guide RNA can comprise the nucleotide sequence: AUUGUUGUAACAUCUAUUUUGUAAGGUGUAAACAAC (SEQ ID NO:70; see FIG. 2KK).

The reverse complement of any one of the nucleotide sequences depicted in FIGS. 2A-2Z, FIGS. 2AA-2ZZ, and FIGS. 2AAA-2FFF (but with T substituted with U) can be included in a guide RNA comprising constant region-(N)n, where N is any nucleotide and n is an integer from 15 to 50 (e.g., from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 35, from 35 to 38, from 35 to 40, from 40 to 45, or from 45 to 50). As one example, a guide RNA can have the following nucleotide sequence: NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAUUGUUGUAACUCUUAUUU UGUAUGGAGUAAACAAC (SEQ ID NO:71) or in some cases the reverse complement, where N is any nucleotide, e.g., where the stretch of Ns includes a target nucleic acid-binding sequence.

Cas12L Guide Polynucleotides

In some cases, a nucleic acid that binds to a Cas12L protein, forming a nucleic acid/Cas12L polypeptide complex, and that targets the complex to a specific location within a target nucleic acid (e.g., a target DNA) comprises ribonucleotides only, deoxyribonucleotides only, or a mixture of ribonucleotides and deoxyribonucleotides. In some cases, a guide polynucleotide comprises ribonucleotides only, and is referred to herein as a “guide RNA.” In some cases, a guide polynucleotide comprises deoxyribonucleotides only, and is referred to herein as a “guide DNA.” In some cases, a guide polynucleotide comprises both ribonucleotides and deoxyribonucleotides. A guide polynucleotide can comprise combinations of ribonucleotide bases, deoxyribonucleotide bases, nucleotide analogs, modified nucleotides, and the like; and may further include naturally-occurring backbone residues and/or linkages and/or non-naturally-occurring backbone residues and/or linkages.

Cas12L Systems

The present disclosure provides a Cas12L system. A Cas12L system of the present disclosure can comprise: a) a Cas12L polypeptide of the present disclosure and a Cas12L guide RNA; b) a Cas12L polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; c) a Cas12L fusion polypeptide of the present disclosure and a Cas12L guide RNA; d) a Cas12L fusion polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; e) an mRNA encoding a Cas12L polypeptide of the present disclosure; and a Cas12L guide RNA; f) an mRNA encoding a Cas12L polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; g) an mRNA encoding a Cas12L fusion polypeptide of the present disclosure; and a Cas12L guide RNA; h) an mRNA encoding a Cas12L fusion polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; i) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure and a nucleotide sequence encoding a Cas12L guide RNA; j) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, a nucleotide sequence encoding a Cas12L guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; k) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure and a nucleotide sequence encoding a Cas12L guide RNA; 1) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, a nucleotide sequence encoding a Cas12L guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; m) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; n) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; and a donor template nucleic acid; o) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; p) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; and a donor template nucleic acid; q) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, a nucleotide sequence encoding a first Cas12L guide RNA, and a nucleotide sequence encoding a second Cas12L guide RNA; or r) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, a nucleotide sequence encoding a first Cas12L guide RNA, and a nucleotide sequence encoding a second Cas12L guide RNA; or some variation of one of (a) through (r).

Nucleic Acids

The present disclosure provides one or more nucleic acids comprising one or more of: a donor polynucleotide sequence, a nucleotide sequence encoding a Cas12L polypeptide (e.g., a wild type Cas12L protein, a nickase Cas12L protein, a dCas12L protein, fusion Cas12L protein, and the like), a Cas12L guide RNA, and a nucleotide sequence encoding a Cas12L guide RNA. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a Cas12L fusion polypeptide. The present disclosure provides a recombinant expression vector that comprises a nucleotide sequence encoding a Cas12L polypeptide. The present disclosure provides a recombinant expression vector that comprises a nucleotide sequence encoding a Cas12L fusion polypeptide. The present disclosure provides a recombinant expression vector that comprises: a) a nucleotide sequence encoding a Cas12L polypeptide; and b) a nucleotide sequence encoding a Cas12L guide RNA(s). The present disclosure provides a recombinant expression vector that comprises: a) a nucleotide sequence encoding a Cas12L fusion polypeptide; and b) a nucleotide sequence encoding a Cas12L guide RNA(s). In some cases, the nucleotide sequence encoding the Cas12L protein and/or the nucleotide sequence encoding the Cas12L guide RNA is operably linked to a promoter that is operable in a cell type of choice (e.g., a prokaryotic cell, a eukaryotic cell, a plant cell, an animal cell, a mammalian cell, a primate cell, a rodent cell, a human cell, etc.).

In some cases, a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure is codon optimized. This type of optimization can entail a mutation of a Cas12L-encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same protein. Thus, the codons can be changed, but the encoded protein remains unchanged. For example, if the intended target cell was a human cell, a human codon-optimized Cas12L-encoding nucleotide sequence could be used. As another non-limiting example, if the intended host cell were a mouse cell, then a mouse codon-optimized Cas12L-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a plant cell, then a plant codon-optimized Cas12L-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were an insect cell, then an insect codon-optimized Cas12L-encoding nucleotide sequence could be generated.

Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www[dot]kazusa[dot]or[dot]jp[forwardslash]codon. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a eukaryotic cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in an animal cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a fungus cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a plant cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a monocotyledonous plant species. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a dicotyledonous plant species. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a gymnosperm plant species. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in an angiosperm plant species. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a corn cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a soybean cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a rice cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a wheat cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a cotton cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a sorghum cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in an alfalfa cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a sugar cane cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in an Arabidopsis cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a tomato cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a cucumber cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in a potato cell. In some cases, a nucleic acid of the present disclosure comprises a Cas12L polypeptide-encoding nucleotide sequence that is codon optimized for expression in an algae cell.

The present disclosure provides one or more recombinant expression vectors that include (in different recombinant expression vectors in some cases, and in the same recombinant expression vector in some cases): (i) a nucleotide sequence of a donor template nucleic acid (where the donor template comprises a nucleotide sequence having homology to a target sequence of a target nucleic acid (e.g., a target genome)); (ii) a nucleotide sequence that encodes a Cas12L guide RNA that hybridizes to a target sequence of the target locus of the targeted genome (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell); and (iii) a nucleotide sequence encoding a Cas12L protein (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell). The present disclosure provides one or more recombinant expression vectors that include (in different recombinant expression vectors in some cases, and in the same recombinant expression vector in some cases): (i) a nucleotide sequence of a donor template nucleic acid (where the donor template comprises a nucleotide sequence having homology to a target sequence of a target nucleic acid (e.g., a target genome)); and (ii) a nucleotide sequence that encodes a Cas12L guide RNA that hybridizes to a target sequence of the target locus of the targeted genome (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell). The present disclosure provides one or more recombinant expression vectors that include (in different recombinant expression vectors in some cases, and in the same recombinant expression vector in some cases): (i) a nucleotide sequence that encodes a Cas12L guide RNA that hybridizes to a target sequence of the target locus of the targeted genome (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell); and (ii) a nucleotide sequence encoding a Cas12L protein (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell).

Suitable expression vectors include viral expression vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. In some cases, a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. In some cases, a recombinant expression vector of the present disclosure is a recombinant lentivirus vector. In some cases, a recombinant expression vector of the present disclosure is a recombinant retroviral vector.

For plant applications, viral vectors based on Tobamoviruses, Potexviruses, Potyviruses, Tobraviruses, Tombusviruses, Geminiviruses, Bromoviruses, Carmoviruses, Alfamoviruses, or Cucumoviruses can be used. See, e.g., Peyret and Lomonossoff (2015) Plant Biotechnol. J. 13:1121. Suitable Tobamovirus vectors include, for example, a tomato mosaic virus (ToMV) vector, a tobacco mosaic virus (TMV) vector, a tobacco mild green mosaic virus (TMGMV) vector, a pepper mild mottle virus (PMMoV) vector, a paprika mild mottle virus (PaMMV) vector, a cucumber green mottle mosaic virus (CGMMV) vector, a kyuri green mottle mosaic virus (KGMMV) vector, a hibiscus latent fort pierce virus (HLFPV) vector, an odontoglossum ringspot virus (ORSV) vector, a rehmannia mosaic virus (ReMV) vector, a Sammon's opuntia virus (SOV) vector, a wasabi mottle virus (WMoV) vector, a youcai mosaic virus (YoMV) vector, a sunn-hemp mosaic virus (SHMV) vector, and the like. Suitable Potexvirus vectors include, for example, a potato virus X (PVX) vector, a potato aucubamosaicvirus (PAMV) vector, an Alstroemeria virus X (AlsVX) vector, a cactus virus X (CVX) vector, a Cymbidium mosaic virus (CymMV) vector, a hosta virus X (HVX) vector, a lily virus X (LVX) vector, a Narcissus mosaic virus (NMV) vector, a Nerine virus X (NVX) vector, a Plantago asiatica mosaic virus (PlAMV) vector, a strawberry mild yellow edge virus (SMYEV) vector, a tulip virus X (TVX) vector, a white clover mosaic virus (WClMV) vector, a bamboo mosaic virus (BaMV) vector, and the like. Suitable Potyvirus vectors include, for example, a potato virus Y (PVY) vector, a bean common mosaic virus (BCMV) vector, a clover yellow vein virus (ClYVV) vector, an East Asian Passiflora virus (EAPV) vector, a Freesia mosaic virus (FreMV) vector, a Japanese yam mosaic virus (JYMV) vector, a lettuce mosaic virus (LMV) vector, a Maize dwarf mosaic virus (MDMV) vector, an onion yellow dwarf virus (OYDV) vector, a papaya ringspot virus (PRSV) vector, a pepper mottle virus (PepMoV) vector, a Perilla mottle virus (PerMoV) vector, a plum pox virus (PPV) vector, a potato virus A (PVA) vector, a sorghum mosaic virus (SrMV) vector, a soybean mosaic virus (SMV) vector, a sugarcane mosaic virus (SCMV) vector, a tulip mosaic virus (TulMV) vector, a turnip mosaic virus (TuMV) vector, a watermelon mosaic virus (WMV) vector, a zucchini yellow mosaic virus (ZYMV) vector, a tobacco etch virus (TEV) vector, and the like. Suitable Tobravirus vectors include, for example, a tobacco rattle virus (TRV) vector and the like. Suitable Tombusvirus vectors include, for example, a tomato bushy stunt virus (TBSV) vector, an eggplant mottled crinkle virus (EMCV) vector, a grapevine Algerian latent virus (GALV) vector, and the like. Suitable Cucumovirus vectors include, for example, a cucumber mosaic virus (CMV) vector, a peanut stunt virus (PSV) vector, a tomato aspermy virus (TAV) vector, and the like. Suitable Bromovirus vectors include, for example, a brome mosaic virus (BMV) vector, a cowpea chlorotic mottle virus (CCMV) vector, and the like. Suitable Carmovirus vectors include, for example, a carnation mottle virus (CarMV) vector, a melon necrotic spot virus (MNSV) vector, a pea stem necrotic virus (PSNV) vector, a turnip crinkle virus (TCV) vector, and the like. Suitable Alfamovirus vectors include, for example, an alfalfa mosaic virus (AMV) vector, and the like.

Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.

In some embodiments, a nucleotide sequence encoding a Cas12L guide RNA is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. In some embodiments, a nucleotide sequence encoding a Cas12L protein or a Cas12L fusion polypeptide is operably linked to a control element, e.g., a transcriptional control element, such as a promoter.

The transcriptional control element can be a promoter. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatable promoter. In some cases, the promoter is an inducible promoter. In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell type-specific promoter. In some cases, the transcriptional control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population. For example, in some cases, the transcriptional control element can be functional in eukaryotic cells, e.g., hematopoietic stem cells (e.g., mobilized peripheral blood (mPB) CD34(+) cell, bone marrow (BM) CD34(+) cell, etc.).

Non-limiting examples of eukaryotic promoters (promoters functional in a eukaryotic cell) include EF1α, those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include nucleotide sequences encoding protein tags (e.g., 6×His tag, hemagglutinin tag, fluorescent protein, etc.) that can be fused to the Cas12L protein, thus resulting in a fusion Cas12L polypeptide.

In some embodiments, a nucleotide sequence encoding a Cas12L guide RNA and/or a Cas12L fusion polypeptide is operably linked to an inducible promoter. In some embodiments, a nucleotide sequence encoding a Cas12L guide RNA and/or a Cas12L fusion protein is operably linked to a constitutive promoter.

A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active/“ON” state), it may be an inducible promoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.), it may be a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).

Suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1;31(17)), a human H1 promoter (H1), and the like.

In some cases, a nucleotide sequence encoding a Cas12L guide RNA is operably linked to (under the control of) a promoter operable in a eukaryotic cell (e.g., a U6 promoter, an enhanced U6 promoter, an H1 promoter, and the like). As would be understood by one of ordinary skill in the art, when expressing an RNA (e.g., a guide RNA) from a nucleic acid (e.g., an expression vector) using a U6 promoter (e.g., in a eukaryotic cell), or another PolIII promoter, the RNA may need to be mutated if there are several Ts in a row (coding for Us in the RNA). This is because a string of Ts (e.g., 5 Ts) in DNA can act as a terminator for polymerase III (PolIII). Thus, in order to ensure transcription of a guide RNA in a eukaryotic cell it may sometimes be necessary to modify the sequence encoding the guide RNA to eliminate runs of Ts. In some cases, a nucleotide sequence encoding a Cas12L protein (e.g., a wild type Cas12L protein, a nickase Cas12L protein, a dCas12L protein, a fusion Cas12L protein and the like) is operably linked to a promoter operable in a eukaryotic cell (e.g., a CMV promoter, an EF1α promoter, an estrogen receptor-regulated promoter, and the like).

Examples of inducible promoters include, but are not limited to T7 RNA polymerase promoter, T3 RNA polymerase promoter, Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose induced promoter, heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc. Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; estrogen and/or an estrogen analog; IPTG; etc.

Inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

In some cases, the promoter is a spatially restricted promoter (i.e., cell type specific promoter, tissue specific promoter, etc.) such that in a multi-cellular organism, the promoter is active (i.e., “ON”) in a subset of specific cells. Spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell; prokaryotic cell).

In some cases, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

RNA polymerase III (Pol III) promoters can be used to drive the expression of non-protein coding RNA molecules (e.g., guide RNAs). In some cases, a suitable promoter is a Pol III promoter. In some cases, a Pol III promoter is operably linked to a nucleotide sequence encoding a guide RNA (gRNA). In some cases, a Pol III promoter is operably linked to a nucleotide sequence encoding a single-guide RNA (sgRNA). In some cases, a Pol III promoter is operably linked to a nucleotide sequence encoding a CRISPR RNA (crRNA). In some cases, a Pol III promoter is operably linked to a nucleotide sequence encoding a encoding a tracrRNA.

Non-limiting examples of Pol III promoters include a U6 promoter, an H1 promoter, a 5S promoter, an Adenovirus 2 (Ad2) VAI promoter, a tRNA promoter, and a 7SK promoter. See, for example, Schramm and Hernandez (2002) Genes & Development 16:2593-2620. In some cases, a Pol III promoter is selected from the group consisting of a U6 promoter, an H1 promoter, a 5S promoter, an Adenovirus 2 (Ad2) VAI promoter, a tRNA promoter, and a 7SK promoter. In some cases, a guide RNA-encoding nucleotide sequence is operably linked to a promoter selected from the group consisting of a U6 promoter, an H1 promoter, a 5S promoter, an Adenovirus 2 (Ad2) VAI promoter, a tRNA promoter, and a 7SK promoter. In some cases, a single-guide RNA-encoding nucleotide sequence is operably linked to a promoter selected from the group consisting of a U6 promoter, an H1 promoter, a 5S promoter, an Adenovirus 2 (Ad2) VAI promoter, a tRNA promoter, and a 7SK promoter.

Examples describing a promoter that can be used herein in connection with expression in plants, plant tissues, and plant cells include, but are not limited to, promoters described in: U.S. Pat. No. 6,437,217 (maize RS81 promoter), U.S. Pat. No. 5,641,876 (rice actin promoter), U.S. Pat. No. 6,426,446 (maize RS324 promoter), U.S. Pat. No. 6,429,362 (maize PR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter), U.S. Pat. No. 6,177,611 (constitutive maize promoters), U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Pat. No. 6,433,252 (maize L3 oleosin promoter), U.S. Pat. No. 6,429,357 (rice actin 2 promoter as well as a rice actin 2 intron), U.S. Pat. No. 5,837,848 (root specific promoter), U.S. Pat. No. 6,294,714 (light inducible promoters), U.S. Pat. No. 6,140,078 (salt inducible promoters), U.S. Pat. No. 6,252,138 (pathogen inducible promoters), U.S. Pat. No. 6,175,060 (phosphorus deficiency inducible promoters), U.S. Pat. No. 6,635,806 (gamma-coixin promoter), and U.S. patent application Ser. No. 09/757,089 (maize chloroplast aldolase promoter). Additional promoters that can find use include a nopaline synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (which is carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al. Plant Molecular Biology (1987) 9: 315-324), the CaMV 35S promoter (Odell et al., Nature (1985) 313: 810-812), the figwort mosaic virus 35S-promoter (U.S. Pat. Nos. 6,051,753; 5,378,619), the sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA (1990) 87: 4144-4148), the R gene complex promoter (Chandler et al., Plant Cell (1989) 1: 1175-1183), and the chlorophyll a/b binding protein gene promoter, PC1SV (U.S. Pat. No. 5,850,019), and AGRtu.nos (GenBank Accession V00087; Depicker et al., Journal of Molecular and Applied Genetics (1982) 1: 561-573; Bevan et al., 1983) promoters.

Methods of introducing a nucleic acid (e.g., a nucleic acid comprising a donor polynucleotide sequence, one or more nucleic acids encoding a Cas12L protein and/or a Cas12L guide RNA, and the like) into a host cell are known in the art, and any convenient method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include e.g., viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

Introducing the recombinant expression vector into cells can occur in any culture media and under any culture conditions that promote the survival of the cells. Introducing the recombinant expression vector into a target cell can be carried out in vivo or ex vivo. Introducing the recombinant expression vector into a target cell can be carried out in vitro.

In some embodiments, a Cas12L protein can be provided as RNA. The RNA can be provided by direct chemical synthesis or may be transcribed in vitro from a DNA (e.g., encoding the Cas12L protein). Once synthesized, the RNA may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).

Nucleic acids may be provided to the cells using well-developed transfection techniques; see, e.g. Angel and Yanik (2010) PLoS ONE 5(7): el1756, and the commercially available TransMessenger® reagents from Qiagen, StemfectT™ RNA Transfection Kit from Stemgent, and TransIT®-mRNA Transfection Kit from Mirus Bio LLC. See also Beumer et al. (2008) PNAS 105(50):19821-19826.

Vectors may be provided directly to a target host cell. In other words, the cells are contacted with vectors comprising the subject nucleic acids (e.g., recombinant expression vectors having the donor template sequence and encoding the Cas12L guide RNA; recombinant expression vectors encoding the Cas12L protein; etc.) such that the vectors are taken up by the cells. Methods for contacting cells with nucleic acid vectors that are plasmids, include electroporation, calcium chloride transfection, microinjection, and lipofection are well known in the art. For viral vector delivery, cells can be contacted with viral particles comprising the subject viral expression vectors.

Retroviruses, for example, lentiviruses, are suitable for use in methods of the present disclosure. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art. Nucleic acids can also introduced by direct micro-injection (e.g., injection of RNA).

Vectors used for providing the nucleic acids encoding Cas12L guide RNA and/or a Cas12L polypeptide to a target host cell can include suitable promoters for driving the expression, that is, transcriptional activation, of the nucleic acid of interest. In other words, in some cases, the nucleic acid of interest will be operably linked to a promoter. This may include ubiquitously acting promoters, for example, the CMV-3-actin promoter, or inducible promoters, such as promoters that are active in particular cell populations or that respond to the presence of drugs such as tetracycline. By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by 10 fold, by 100 fold, more usually by 1000 fold. In addition, vectors used for providing a nucleic acid encoding a Cas12L guide RNA and/or a Cas12L protein to a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the Cas12L guide RNA and/or Cas12L protein.

A nucleic acid comprising a nucleotide sequence encoding a Cas12L polypeptide, or a Cas12L fusion polypeptide, is in some cases an RNA. Thus, a Cas12L fusion protein can be introduced into cells as RNA. Methods of introducing RNA into cells are known in the art and may include, for example, direct injection, transfection, or any other method used for the introduction of DNA. A Cas12L protein may instead be provided to cells as a polypeptide. Such a polypeptide may optionally be fused to a polypeptide domain that increases solubility of the product. The domain may be linked to the polypeptide through a defined protease cleavage site, e.g. a TEV sequence, which is cleaved by TEV protease. The linker may also include one or more flexible sequences, e.g. from 1 to 10 glycine residues. In some embodiments, the cleavage of the fusion protein is performed in a buffer that maintains solubility of the product, e.g. in the presence of from 0.5 to 2 M urea, in the presence of polypeptides and/or polynucleotides that increase solubility, and the like. Domains of interest include endosomolytic domains, e.g. influenza HA domain; and other polypeptides that aid in production, e.g. IF2 domain, GST domain, GRPE domain, and the like. The polypeptide may be formulated for improved stability. For example, the peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream.

Additionally or alternatively, a Cas12L polypeptide of the present disclosure may be fused to a polypeptide permeant domain to promote uptake by the cell. A number of permeant domains are known in the art and may be used in the non-integrating polypeptides of the present disclosure, including peptides, peptidomimetics, and non-peptide carriers. For example, a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO:44). As another example, the permeant peptide comprises the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally-occurring tat protein. Other permeant domains include poly-arginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nona-arginine, octa-arginine, and the like. (See, for example, Futaki et al. (2003) Curr Protein Pept Sci. 2003 April; 4(2): 87-9 and 446; and Wender et al. (2000) Proc. Natl. Acad. Sci. U.S.A 2000 Nov. 21; 97(24):13003-8; published U.S. patent applications 20030220334; 20030083256; 20030032593; and 20030022831, herein specifically incorporated by reference for the teachings of translocation peptides and peptoids). The nona-arginine (R9) sequence is one of the more efficient PTDs that have been characterized (Wender et al. 2000; Uemura et al. 2002). The site at which the fusion is made may be selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide. The optimal site will be determined by routine experimentation.

As noted above, in some cases, the target cell is a plant cell. Numerous methods for transforming chromosomes or plastids in a plant cell with a recombinant nucleic acid are known in the art, which can be used according to methods of the present application to produce a transgenic plant cell and/or a transgenic plant. Any suitable method or technique for transformation of a plant cell known in the art can be used. Effective methods for transformation of plants include bacterially mediated transformation, such as Agrobacterium-mediated or Rhizobium-mediated transformation and microprojectile bombardment-mediated transformation. A variety of methods are known in the art for transforming explants with a transformation vector via bacterially mediated transformation or microprojectile bombardment and then subsequently culturing, etc., those explants to regenerate or develop transgenic plants. Other methods for plant transformation, such as microinjection, electroporation, vacuum infiltration, pressure, sonication, silicon carbide fiber agitation, PEG-mediated transformation, etc., are also known in the art. Transgenic plants produced by these transformation methods can be chimeric or non-chimeric for the transformation event depending on the methods and explants used.

Methods of transforming plant cells are well known by persons of ordinary skill in the art. For instance, specific instructions for transforming plant cells by microprojectile bombardment with particles coated with recombinant DNA (e.g., biolistic transformation) are found in U.S. Pat. Nos. 5,550,318; 5,538,880 6,160,208; 6,399,861; and 6,153,812 and Agrobacterium-mediated transformation is described in U.S. Pat. Nos. 5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871; 5,463,174; and 5,188,958. Additional methods for transforming plants can be found in, for example, Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Any appropriate method known to those skilled in the art can be used to transform a plant cell with any of the nucleic acids provided herein.

A Cas12L polypeptide of the present disclosure may be produced in vitro or by eukaryotic cells or by prokaryotic cells, and it may be further processed by unfolding, e.g. heat denaturation, dithiothreitol reduction, etc. and may be further refolded, using methods known in the art.

Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

Also suitable for inclusion in embodiments of the present disclosure are nucleic acids (e.g., encoding a Cas12L guide RNA, encoding a Cas12L fusion protein, etc.) and proteins (e.g., a Cas12L fusion protein derived from a wild type protein or a variant protein) that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation, to change the target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., transcription modulatory activity, enzymatic activity, etc.) or to render them more suitable. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.

A Cas12L polypeptide of the present disclosure may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

A Cas12L polypeptide of the present disclosure may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise 20% or more by weight of the desired product, more usually 75% or more by weight, preferably 95% or more by weight, and for therapeutic purposes, usually 99.5% or more by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein. Thus, in some cases, a Cas12L polypeptide, or a Cas12L fusion polypeptide, of the present disclosure is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-Cas12L proteins or other macromolecules, etc.).

To induce cleavage or any desired modification to a target nucleic acid (e.g., genomic DNA), or any desired modification to a polypeptide associated with target nucleic acid, the Cas12L guide RNA and/or the Cas12L polypeptide of the present disclosure and/or the donor template sequence, whether they be introduced as nucleic acids or polypeptides, are provided to the cells for about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about 30 minutes to about 24 hours, which may be repeated with a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days. The agent(s) may be provided to the subject cells one or more times, e.g. one time, twice, three times, or more than three times, and the cells allowed to incubate with the agent(s) for some amount of time following each contacting event e.g. 16-24 hours, after which time the media is replaced with fresh media and the cells are cultured further.

In cases in which two or more different targeting complexes are provided to the cell (e.g., two different Cas12L guide RNAs that are complementary to different sequences within the same or different target nucleic acid), the complexes may be provided simultaneously (e.g. as two polypeptides and/or nucleic acids), or delivered simultaneously. Alternatively, they may be provided consecutively, e.g. the targeting complex being provided first, followed by the second targeting complex, etc. or vice versa.

To improve the delivery of a DNA vector into a target cell, the DNA can be protected from damage and its entry into the cell facilitated, for example, by using lipoplexes and polyplexes. Thus, in some cases, a nucleic acid of the present disclosure (e.g., a recombinant expression vector of the present disclosure) can be covered with lipids in an organized structure like a micelle or a liposome. When the organized structure is complexed with DNA it is called a lipoplex. There are three types of lipids, anionic (negatively-charged), neutral, or cationic (positively-charged). Lipoplexes that utilize cationic lipids have proven utility for gene transfer. Cationic lipids, due to their positive charge, naturally complex with the negatively charged DNA. Also as a result of their charge, they interact with the cell membrane. Endocytosis of the lipoplex then occurs, and the DNA is released into the cytoplasm. The cationic lipids also protect against degradation of the DNA by the cell.

Complexes of polymers with DNA are called polyplexes. Most polyplexes consist of cationic polymers and their production is regulated by ionic interactions. One large difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA load into the cytoplasm, so to this end, co-transfection with endosome-lytic agents (to lyse the endosome that is made during endocytosis) such as inactivated adenovirus must occur. However, this is not always the case; polymers such as polyethylenimine have their own method of endosome disruption as does chitosan and trimethylchitosan.

Dendrimers, a highly branched macromolecule with a spherical shape, may be also be used to genetically modify stem cells. The surface of the dendrimer particle may be functionalized to alter its properties. In particular, it is possible to construct a cationic dendrimer (i.e., one with a positive surface charge). When in the presence of genetic material such as a DNA plasmid, charge complementarity leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching its destination, the dendrimer-nucleic acid complex can be taken up into a cell by endocytosis.

In some cases, a nucleic acid of the disclosure (e.g., an expression vector) includes an insertion site for a guide sequence of interest. For example, a nucleic acid can include an insertion site for a guide sequence of interest, where the insertion site is immediately adjacent to a nucleotide sequence encoding the portion of a Cas12L guide RNA that does not change when the guide sequence is changed to hybridized to a desired target sequence (e.g., sequences that contribute to the Cas12L binding aspect of the guide RNA, e.g., the sequences that contribute to the dsRNA duplex(es) of the Cas12L guide RNA—this portion of the guide RNA can also be referred to as the ‘scaffold’ or ‘constant region’ of the guide RNA). Thus, in some cases, a subject nucleic acid (e.g., an expression vector) includes a nucleotide sequence encoding a Cas12L guide RNA, except that the portion encoding the guide sequence portion of the guide RNA is an insertion sequence (an insertion site). An insertion site is any nucleotide sequence used for the insertion of the desired sequence. “Insertion sites” for use with various technologies are known to those of ordinary skill in the art and any convenient insertion site can be used. An insertion site can be for any method for manipulating nucleic acid sequences. For example, in some cases the insertion site is a multiple cloning site (MCS) (e.g., a site including one or more restriction enzyme recognition sequences), a site for ligation independent cloning, a site for recombination based cloning (e.g., recombination based on att sites), a nucleotide sequence recognized by a CRISPR/Cas (e.g. Cas9) based technology, and the like.

An insertion site can be any desirable length, and can depend on the type of insertion site (e.g., can depend on whether (and how many) the site includes one or more restriction enzyme recognition sequences, whether the site includes a target site for a CRISPR/Cas protein, etc.). In some cases, an insertion site of a subject nucleic acid is 3 or more nucleotides (nt) in length (e.g., 5 or more, 8 or more, 10 or more, 15 or more, 17 or more, 18 or more, 19 or more, 20 or more or 25 or more, or 30 or more nt in length). In some cases, the length of an insertion site of a subject nucleic acid has a length in a range of from 2 to 50 nucleotides (nt) (e.g., from 2 to 40 nt, from 2 to 30 nt, from 2 to 25 nt, from 2 to 20 nt, from 5 to 50 nt, from 5 to 40 nt, from 5 to 30 nt, from 5 to 25 nt, from 5 to 20 nt, from 10 to 50 nt, from 10 to 40 nt, from 10 to 30 nt, from 10 to 25 nt, from 10 to 20 nt, from 17 to 50 nt, from 17 to 40 nt, from 17 to 30 nt, from 17 to 25 nt). In some cases, the length of an insertion site of a subject nucleic acid has a length in a range of from 5 to 40 nt.

Nucleic Acid Modifications

In some embodiments, a subject nucleic acid (e.g., a Cas12L guide RNA) has one or more modifications, e.g., a base modification, a backbone modification, etc., to provide the nucleic acid with a new or enhanced feature (e.g., improved stability). A nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, the 3′, or the 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are suitable. In addition, linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Suitable nucleic acid modifications include, but are not limited to: 2′Omethyl modified nucleotides, 2′ Fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and a 5′ cap (e.g., a 7-methylguanylate cap (m7G)). Additional details and additional modifications are described below.

A 2′-O-Methyl modified nucleotide (also referred to as 2′-O-Methyl RNA) is a naturally occurring modification of RNA found in tRNA and other small RNAs that arises as a post-transcriptional modification. Oligonucleotides can be directly synthesized that contain 2′-O-Methyl RNA. This modification increases Tm of RNA:RNA duplexes but results in only small changes in RNA:DNA stability. It is stabile with respect to attack by single-stranded ribonucleases and is typically 5 to 10-fold less susceptible to DNases than DNA. It is commonly used in antisense oligos as a means to increase stability and binding affinity to the target message.

2′ Fluoro modified nucleotides (e.g., 2′ Fluoro bases) have a fluorine modified ribose which increases binding affinity (Tm) and also confers some relative nuclease resistance when compared to native RNA. These modifications are commonly employed in ribozymes and siRNAs to improve stability in serum or other biological fluids.

LNA bases have a modification to the ribose backbone that locks the base in the C3′-endo position, which favors RNA A-type helix duplex geometry. This modification significantly increases Tm and is also very nuclease resistant. Multiple LNA insertions can be placed in an oligo at any position except the 3′-end. Applications have been described ranging from antisense oligos to hybridization probes to SNP detection and allele specific PCR. Due to the large increase in Tm conferred by LNAs, they also can cause an increase in primer dimer formation as well as self-hairpin formation. In some cases, the number of LNAs incorporated into a single oligo is 10 bases or less.

The phosphorothioate (PS) bond (i.e., a phosphorothioate linkage) substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of a nucleic acid (e.g., an oligo). This modification renders the internucleotide linkage resistant to nuclease degradation. Phosphorothioate bonds can be introduced between the last 3-5 nucleotides at the 5′- or 3′-end of the oligo to inhibit exonuclease degradation. Including phosphorothioate bonds within the oligo (e.g., throughout the entire oligo) can help reduce attack by endonucleases as well.

In some embodiments, a subject nucleic acid has one or more nucleotides that are 2′-O-Methyl modified nucleotides. In some embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.) has one or more 2′ Fluoro modified nucleotides. In some embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.) has one or more LNA bases. In some embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.) has one or more nucleotides that are linked by a phosphorothioate bond (i.e., the subject nucleic acid has one or more phosphorothioate linkages). In some embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.) has a 5′ cap (e.g., a 7-methylguanylate cap (m7G)). In some embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.) has a combination of modified nucleotides. For example, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.) can have a 5′ cap (e.g., a 7-methylguanylate cap (m7G)) in addition to having one or more nucleotides with other modifications (e.g., a 2′-O-Methyl nucleotide and/or a 2′ Fluoro modified nucleotide and/or a LNA base and/or a phosphorothioate linkage).

Modified Backbones and Modified Internucleoside Linkages

Examples of suitable nucleic acids (e.g., a Cas12L guide RNA) containing modifications include nucleic acids containing modified backbones or non-natural internucleoside linkages. Nucleic acids having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

Suitable modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Suitable oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts (such as, for example, potassium or sodium), mixed salts and free acid forms are also included.

In some embodiments, a subject nucleic acid comprises one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂-(known as a methylene (methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the native phosphodiester internucleotide linkage is represented as —O—P(═O)(OH)—O—CH₂—). MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677, the disclosure of which is incorporated herein by reference in its entirety. Suitable amide internucleoside linkages are disclosed in U.S. Pat. No. 5,602,240, the disclosure of which is incorporated herein by reference in its entirety.

Also suitable are nucleic acids having morpholino backbone structures as described in, e.g., U.S. Pat. No. 5,034,506. For example, in some embodiments, a subject nucleic acid comprises a 6-membered morpholino ring in place of a ribose ring. In some of these embodiments, a phosphorodiamidate or other non-phosphodiester internucleoside linkage replaces a phosphodiester linkage.

Suitable modified polynucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂component parts.

Mimetics

A subject nucleic acid can be a nucleic acid mimetic. The term “mimetic” as it is applied to polynucleotides is intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid, a polynucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA, the sugar-backbone of a polynucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

One polynucleotide mimetic that has been reported to have excellent hybridization properties is a peptide nucleic acid (PNA). The backbone in PNA compounds is two or more linked aminoethylglycine units which gives PNA an amide containing backbone. The heterocyclic base moieties are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that describe the preparation of PNA compounds include, but are not limited to: U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the disclosures of which are incorporated herein by reference in their entirety.

Another class of polynucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. One class of linking groups has been selected to give a non-ionic oligomeric compound. The non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based polynucleotides are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based polynucleotides are disclosed in U.S. Pat. No. 5,034,506, the disclosure of which is incorporated herein by reference in its entirety. A variety of compounds within the morpholino class of polynucleotides have been prepared, having a variety of different linking groups joining the monomeric subunits.

A further class of polynucleotide mimetic is referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in a DNA/RNA molecule is replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602, the disclosure of which is incorporated herein by reference in its entirety). In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation.

A further modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. The linkage can be a methylene (—CH₂—), group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456, the disclosure of which is incorporated herein by reference in its entirety). LNA and LNA analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +100° C.), stability towards 3′-exonucleolytic degradation and good solubility properties. Potent and nontoxic antisense oligonucleotides containing LNAs have been described (e.g., Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638, the disclosure of which is incorporated herein by reference in its entirety).

The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (e.g., Koshkin et al., Tetrahedron, 1998, 54, 3607-3630, the disclosure of which is incorporated herein by reference in its entirety). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226, as well as U.S. applications 20120165514,20100216983,20090041809,20060117410,20040014959,20020094555, and 20020086998, the disclosures of which are incorporated herein by reference in their entirety.

Modified Sugar Moieties

A subject nucleic acid can also include one or more substituted sugar moieties. Suitable polynucleotides comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to C₁₀alkyl or C₂to C₁₀alkenyl and alkynyl. Particularly suitable are O((CH₂)_nO)_mCH₃, O(CH₂)_nOCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON((CH₂)_nCH₃)₂, where n and m are from 1 to about 10. Other suitable polynucleotides comprise a sugar substituent group selected from: C₁to C₁₀lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A suitable modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504, the disclosure of which is incorporated herein by reference in its entirety) i.e., an alkoxyalkoxy group. A further suitable modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O-CH₂—O—CH₂—N(CH₃)₂.

Other suitable sugar substituent groups include methoxy (—O—CH₃), aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. A suitable 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

Base Modifications and Substitutions

A subject nucleic acid may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).

Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993; the disclosures of which are incorporated herein by reference in their entirety. Certain of these nucleobases are useful for increasing the binding affinity of an oligomeric compound. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi et al., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278; the disclosure of which is incorporated herein by reference in its entirety) and are suitable base substitutions, e.g., when combined with 2′-O-methoxyethyl sugar modifications.

Conjugates

Another possible modification of a subject nucleic acid involves chemically linking to the polynucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a subject nucleic acid.

Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

A conjugate may include a “Protein Transduction Domain” or PTD (also known as a CPP-cell penetrating peptide), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from a small polar molecule to a large macromolecule and/or a nanoparticle, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle (e.g., the nucleus). In some embodiments, a PTD is covalently linked to the 3′ end of an exogenous polynucleotide. In some embodiments, a PTD is covalently linked to the 5′ end of an exogenous polynucleotide. Exemplary PTDs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; SEQ ID NO:40); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natd. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:41); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:42); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:43); and RQIKIWFQNRRMKWKK (SEQ ID NO:44). Exemplary PTDs include but are not limited to, YGRKKRRQRRR (SEQ ID NO:40), RKKRRQRRR (SEQ ID NO:45); an arginine homopolymer of from 3 arginine residues to 50 arginine residues; Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR SEQ ID NO:40); RKKRRQRR (SEQ ID NO:46); YARAAARQARA SEQ ID NO:47); THRLPRRRRRR (SEQ ID NO:48); and GGRRARRRRRR (SEQ ID NO:49). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”) connected via a cleavable linker to a matching polyanion (e.g., Glu9 or “E9”), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus “activating” the ACPP to traverse the membrane.

Introducing Components into a Target Cell

A Cas12L guide RNA (or a nucleic acid comprising a nucleotide sequence encoding same) and/or a Cas12L polypeptide of the present disclosure (or a nucleic acid comprising a nucleotide sequence encoding same) and/or a Cas12L fusion polypeptide of the present disclosure (or a nucleic acid that includes a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure) and/or a donor polynucleotide (donor template) can be introduced into a host cell by any of a variety of well-known methods.

Any of a variety of compounds and methods can be used to deliver to a target cell a Cas12L system of the present disclosure (e.g., where a Cas12L system comprises: a) a Cas12L polypeptide of the present disclosure and a Cas12L guide RNA; b) a Cas12L polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; c) a Cas12L fusion polypeptide of the present disclosure and a Cas12L guide RNA; d) a Cas12L fusion polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; e) an mRNA encoding a Cas12L polypeptide of the present disclosure; and a Cas12L guide RNA; f) an mRNA encoding a Cas12L polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; g) an mRNA encoding a Cas12L fusion polypeptide of the present disclosure; and a Cas12L guide RNA; h) an mRNA encoding a Cas12L fusion polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; i) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure and a nucleotide sequence encoding a Cas12L guide RNA; j) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, a nucleotide sequence encoding a Cas12L guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; k) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure and a nucleotide sequence encoding a Cas12L guide RNA; 1) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, a nucleotide sequence encoding a Cas12L guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; m) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; n) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; and a donor template nucleic acid; o) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; p) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; and a donor template nucleic acid; q) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, a nucleotide sequence encoding a first Cas12L guide RNA, and a nucleotide sequence encoding a second Cas12L guide RNA; or r) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, a nucleotide sequence encoding a first Cas12L guide RNA, and a nucleotide sequence encoding a second Cas12L guide RNA; or some variation of one of (a) through (r). As a non-limiting example, a Cas12L system of the present disclosure can be combined with a lipid. As another non-limiting example, a Cas12L system of the present disclosure can be combined with a particle, or formulated into a particle.

Methods of introducing a nucleic acid into a host cell are known in the art, and any convenient method can be used to introduce a subject nucleic acid (e.g., an expression construct/vector) into a target cell (e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, and the like). Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et., al Adv Drug Deliv Rev. 2012 Sep. 13. pii: 50169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023), and the like.

In some cases, a Cas12L polypeptide of the present disclosure is provided as a nucleic acid (e.g., an mRNA, a DNA, a plasmid, an expression vector, a viral vector, etc.) that encodes the Cas12L polypeptide. In some cases, the Cas12L polypeptide of the present disclosure is provided directly as a protein (e.g., without an associated guide RNA or with an associate guide RNA, i.e., as a ribonucleoprotein complex). A Cas12L polypeptide of the present disclosure can be introduced into a cell (provided to the cell) by any convenient method; such methods are known to those of ordinary skill in the art. As an illustrative example, a Cas12L polypeptide of the present disclosure can be injected directly into a cell (e.g., with or without a Cas12L guide RNA or nucleic acid encoding a Cas12L guide RNA, and with or without a donor polynucleotide). As another example, a preformed complex of a Cas12L polypeptide of the present disclosure and a Cas12L guide RNA (an RNP) can be introduced into a cell (e.g, eukaryotic cell) (e.g., via injection, via nucleofection; via a protein transduction domain (PTD) conjugated to one or more components, e.g., conjugated to the Cas12L protein, conjugated to a guide RNA, conjugated to a Cas12L polypeptide of the present disclosure and a guide RNA; etc.).

In some cases, a Cas12L fusion polypeptide (e.g., dCas12L fused to a fusion partner, nickase Cas12L fused to a fusion partner, etc.) of the present disclosure is provided as a nucleic acid (e.g., an mRNA, a DNA, a plasmid, an expression vector, a viral vector, etc.) that encodes the Cas12L fusion polypeptide. In some cases, the Cas12L fusion polypeptide of the present disclosure is provided directly as a protein (e.g., without an associated guide RNA or with an associate guide RNA, i.e., as a ribonucleoprotein complex). A Cas12L fusion polypeptide of the present disclosure can be introduced into a cell (provided to the cell) by any convenient method; such methods are known to those of ordinary skill in the art. As an illustrative example, a Cas12L fusion polypeptide of the present disclosure can be injected directly into a cell (e.g., with or without nucleic acid encoding a Cas12L guide RNA and with or without a donor polynucleotide). As another example, a preformed complex of a Cas12L fusion polypeptide of the present disclosure and a Cas12L guide RNA (an RNP) can be introduced into a cell (e.g., via injection, via nucleofection; via a protein transduction domain (PTD) conjugated to one or more components, e.g., conjugated to the Cas12L fusion protein, conjugated to a guide RNA, conjugated to a Cas12L fusion polypeptide of the present disclosure and a guide RNA; etc.).

In some cases, a nucleic acid (e.g., a Cas12L guide RNA; a nucleic acid comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure; etc.) is delivered to a cell (e.g., a target host cell) and/or a polypeptide (e.g., a Cas12L polypeptide; a Cas12L fusion polypeptide) in a particle, or associated with a particle. In some cases, a Cas12L system of the present disclosure is delivered to a cell in a particle, or associated with a particle. The terms “particle” and nanoparticle” can be used interchangeable, as appropriate. A recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure and/or a Cas12L guide RNA, an mRNA comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, and guide RNA may be delivered simultaneously using particles or lipid envelopes; for instance, a Cas12L polypeptide and a Cas12L guide RNA, e.g., as a complex (e.g., a ribonucleoprotein (RNP) complex), can be delivered via a particle, e.g., a delivery particle comprising lipid or lipidoid and hydrophilic polymer, e.g., a cationic lipid and a hydrophilic polymer, for instance wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or wherein the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and/or wherein the particle further comprises cholesterol (e.g., particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0; formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5). For example, a particle can be formed using a multistep process in which a Cas12L polypeptide and a Cas12L guideRNA are mixed together, e.g., at a 1:1 molar ratio, e.g., at room temperature, e.g., for 30 minutes, e.g., in sterile, nuclease free 1×phosphate-buffered saline (PBS); and separately, DOTAP, DMPC, PEG, and cholesterol as applicable for the formulation are dissolved in alcohol, e.g., 100% ethanol; and, the two solutions are mixed together to form particles containing the complexes).

A Cas12L polypeptide of the present disclosure (or an mRNA comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure; or a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure) and/or Cas12L guide RNA (or a nucleic acid such as one or more expression vectors encoding the Cas12L guide RNA) may be delivered simultaneously using particles or lipid envelopes. For example, a biodegradable core-shell structured nanoparticle with a poly(O-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell can be used. In some cases, particles/nanoparticles based on self assembling bioadhesive polymers are used; such particles/nanoparticles may be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, e.g., to the brain. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs are also contemplated. A molecular envelope technology, which involves an engineered polymer envelope which is protected and delivered to the site of the disease, can be used. Doses of about 5 mg/kg can be used, with single or multiple doses, depending on various factors, e.g., the target tissue.

Lipidoid compounds (e.g., as described in US patent application 20110293703) are also useful in the administration of polynucleotides, and can be used to deliver a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure (e.g., where a Cas12L system comprises: a) a Cas12L polypeptide of the present disclosure and a Cas12L guide RNA; b) a Cas12L polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; c) a Cas12L fusion polypeptide of the present disclosure and a Cas12L guide RNA; d) a Cas12L fusion polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; e) an mRNA encoding a Cas12L polypeptide of the present disclosure; and a Cas12L guide RNA; f) an mRNA encoding a Cas12L polypeptide of the present disclosure, a Cas12L guide RNA, and a donor templat nucleic acid; g) an mRNA encoding a Cas12L fusion polypeptide of the present disclosure; and a Cas12L guide RNA; h) an mRNA encoding a Cas12L fusion polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; i) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure and a nucleotide sequence encoding a Cas12L guide RNA; j) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, a nucleotide sequence encoding a Cas12L guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; k) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure and a nucleotide sequence encoding a Cas12L guide RNA; 1) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, a nucleotide sequence encoding a Cas12L guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; m) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; n) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; and a donor template nucleic acid; o) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; p) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; and a donor template nucleic acid; q) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, a nucleotide sequence encoding a first Cas12L guide RNA, and a nucleotide sequence encoding a second Cas12L guide RNA; or r) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, a nucleotide sequence encoding a first Cas12L guide RNA, and a nucleotide sequence encoding a second Cas12L guide RNA; or some variation of one of (a) through (r). In one aspect, the aminoalcohol lipidoid compounds are combined with an agent to be delivered to a cell or a subject to form microparticles, nanoparticles, liposomes, or micelles. The aminoalcohol lipidoid compounds may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.

A poly(beta-amino alcohol) (PBAA) can be used to deliver a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell. US Patent Publication No. 20130302401 relates to a class of poly(beta-amino alcohols) (PBAAs) that has been prepared using combinatorial polymerization.

Sugar-based particles may be used, for example GalNAc, as described with reference to WO2014118272 (incorporated herein by reference) and Nair, J K et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961) can be used to deliver a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell.

In some cases, lipid nanoparticles (LNPs) are used to deliver a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell. Negatively charged polymers such as RNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times. Four species of ionizable cationic lipids have been focused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). Preparation of LNPs and is described in, e.g., Rosin et al. (2011) Molecular Therapy 19:1286-2200). The cationic lipids 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2″-(methoxypolyethyleneglycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), and R-3-[(.omega.-methoxy-poly(ethylene glycol)2000) carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be used. A nucleic acid (e.g., a Cas12L guide RNA; a nucleic acid of the present disclosure; etc.) may be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios). In some cases, 0.2% SP-DiOC18 is incorporated.

Spherical Nucleic Acid (SNAT™) constructs and other nanoparticles (particularly gold nanoparticles) can be used to deliver a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell.. See, e.g., Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao et al., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970, Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., Nano Lett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012 109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci. USA. 2013 110(19): 7625-7630, Jensen et al., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small, 10:186-192.

Self-assembling nanoparticles with RNA may be constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG).

In general, a “nanoparticle” refers to any particle having a diameter of less than 1000 nm. In some cases, nanoparticles suitable for use in delivering a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell have a diameter of 500 nm or less, e.g., from 25 nm to 35 nm, from 35 nm to 50 nm, from 50 nm to 75 nm, from 75 nm to 100 nm, from 100 nm to 150 nm, from 150 nm to 200 nm, from 200 nm to 300 nm, from 300 nm to 400 nm, or from 400 nm to 500 nm. In some cases, nanoparticles suitable for use in delivering a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell have a diameter of from 25 nm to 200 nm. In some cases, nanoparticles suitable for use in delivering a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell have a diameter of 100 nm or less In some cases, nanoparticles suitable for use in delivering a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell have a diameter of from 35 nm to 60 nm.

Nanoparticles suitable for use in delivering a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell may be provided in different forms, e.g., as solid nanoparticles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof. Metal, dielectric, and semiconductor nanoparticles may be prepared, as well as hybrid structures (e.g., core-shell nanoparticles). Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically below 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present disclosure.

Semi-solid and soft nanoparticles are also suitable for use in delivering a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell. A prototype nanoparticle of semi-solid nature is the liposome.

In some cases, an exosome is used to deliver a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell. Exosomes are endogenous nano-vesicles that transport RNAs and proteins, and which can deliver RNA to the brain and other target organs.

In some cases, a liposome is used to deliver a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes. Although liposome formation is spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus. Several other additives may be added to liposomes in order to modify their structure and properties. For instance, either cholesterol or sphingomyelin may be added to the liposomal mixture in order to help stabilize the liposomal structure and to prevent the leakage of the liposomal inner cargo. A liposome formulation may be mainly comprised of natural phospholipids and lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside.

A stable nucleic-acid-lipid particle (SNALP) can be used to deliver a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell. The SNALP formulation may contain the lipids 3-N-[(methoxypoly(ethylene glycol) 2000) carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a 2:40:10:48 molar percent ratio. The SNALP liposomes may be prepared by formulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulting SNALP liposomes can be about 80-100 nm in size. A SNALP may comprise synthetic cholesterol (Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxy poly(ethylene glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane. A SNALP may comprise synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA).

Other cationic lipids, such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) can be used to deliver a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell. A preformed vesicle with the following lipid composition may be contemplated: amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol and (R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethylene glycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10, respectively, and a FVII siRNA/total lipid ratio of approximately 0.05 (w/w). To ensure a narrow particle size distribution in the range of 70-90 nm and a low polydispersity index of 0.11.+−0.0.04 (n=56), the particles may be extruded up to three times through 80 nm membranes prior to adding the guide RNA. Particles containing the highly potent amino lipid 16 may be used, in which the molar ratio of the four lipid components 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) which may be further optimized to enhance in vivo activity.

Lipids may be formulated with a Cas12L system of the present disclosure or component(s) thereof or nucleic acids encoding the same to form lipid nanoparticles (LNPs). Suitable lipids include, but are not limited to, DLin-KC2-DMA4, C12-200 and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG may be formulated with a Cas12L system, or component thereof, of the present disclosure, using a spontaneous vesicle formation procedure. The component molar ratio may be about 50/10/38.5/1.5 (DLin-KC2-DMA or C12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG).

A Cas12L system of the present disclosure, or a component thereof, may be delivered encapsulated in PLGA microspheres such as that further described in US published applications 20130252281 and 20130245107 and 20130244279.

Supercharged proteins can be used to deliver a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell. Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit the ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, RNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo.

Cell Penetrating Peptides (CPPs) can be used to deliver a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.

An implantable device can be used to deliver a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure (e.g., a Cas12L guide RNA, a nucleic acid encoding a Cas12L guide RNA, a nucleic acid encoding Cas12L polypeptide, a donor template, and the like), or a Cas12L system of the present disclosure, to a target cell (e.g., a target cell in vivo, where the target cell is a target cell in circulation, a target cell in a tissue, a target cell in an organ, etc.). An implantable device suitable for use in delivering a Cas12L polypeptide of the present disclosure, a Cas12L fusion polypeptide of the present disclosure, an RNP of the present disclosure, a nucleic acid of the present disclosure, or a Cas12L system of the present disclosure, to a target cell (e.g., a target cell in vivo, where the target cell is a target cell in circulation, a target cell in a tissue, a target cell in an organ, etc.) can include a container (e.g., a reservoir, a matrix, etc.) that comprises the Cas12L polypeptide, the Cas12L fusion polypeptide, the RNP, or the Cas12L system (or component thereof, e.g., a nucleic acid of the present disclosure).

A suitable implantable device can comprise a polymeric substrate, such as a matrix for example, that is used as the device body, and in some cases additional scaffolding materials, such as metals or additional polymers, and materials to enhance visibility and imaging. An implantable delivery device can be advantageous in providing release locally and over a prolonged period, where the polypeptide and/or nucleic acid to be delivered is released directly to a target site, e.g., the extracellular matrix (ECM), the vasculature surrounding a tumor, a diseased tissue, etc. Suitable implantable delivery devices include devices suitable for use in delivering to a cavity such as the abdominal cavity and/or any other type of administration in which the drug delivery system is not anchored or attached, comprising a biostable and/or degradable and/or bioabsorbable polymeric substrate, which may for example optionally be a matrix. In some cases, a suitable implantable drug delivery device comprises degradable polymers, wherein the main release mechanism is bulk erosion. In some cases, a suitable implantable drug delivery device comprises non degradable, or slowly degraded polymers, wherein the main release mechanism is diffusion rather than bulk erosion, so that the outer part functions as membrane, and its internal part functions as a drug reservoir, which practically is not affected by the surroundings for an extended period (for example from about a week to about a few months). Combinations of different polymers with different release mechanisms may also optionally be used. The concentration gradient at the can be maintained effectively constant during a significant period of the total releasing period, and therefore the diffusion rate is effectively constant (termed “zero mode” diffusion). By the term “constant” it is meant a diffusion rate that is maintained above the lower threshold of therapeutic effectiveness, but which may still optionally feature an initial burst and/or may fluctuate, for example increasing and decreasing to a certain degree. The diffusion rate can be so maintained for a prolonged period, and it can be considered constant to a certain level to optimize the therapeutically effective period, for example the effective silencing period.

In some cases, the implantable delivery system is designed to shield the nucleotide based therapeutic agent from degradation, whether chemical in nature or due to attack from enzymes and other factors in the body of the subject.

The site for implantation of the device, or target site, can be selected for maximum therapeutic efficacy. For example, a delivery device can be implanted within or in the proximity of a tumor environment, or the blood supply associated with a tumor. The target location can be, e.g.: 1) the brain at degenerative sites like in Parkinson or Alzheimer disease at the basal ganglia, white and gray matter; 2) the spine, as in the case of amyotrophic lateral sclerosis (ALS); 3) uterine cervix; 4) active and chronic inflammatory joints; 5) dermis as in the case of psoriasis; 7) sympathetic and sensoric nervous sites for analgesic effect; 7) a bone; 8) a site of acute or chronic infection; 9) Intra vaginal; 10) Inner ear—auditory system, labyrinth of the inner ear, vestibular system; 11) Intra tracheal; 12) Intra-cardiac; coronary, epicardiac; 13) urinary tract or bladder; 14) biliary system; 15) parenchymal tissue including and not limited to the kidney, liver, spleen; 16) lymph nodes; 17) salivary glands; 18) dental gums; 19) Intra-articular (into joints); 20) Intra-ocular; 21) Brain tissue; 22) Brain ventricles; 23) Cavities, including abdominal cavity (for example but without limitation, for ovary cancer); 24) Intra esophageal; and 25) Intra rectal; and 26) into the vasculature.

The method of insertion, such as implantation, may optionally already be used for other types of tissue implantation and/or for insertions and/or for sampling tissues, optionally without modifications, or alternatively optionally only with non-major modifications in such methods. Such methods optionally include but are not limited to brachytherapy methods, biopsy, endoscopy with and/or without ultrasound, such as stereotactic methods into the brain tissue, laparoscopy, including implantation with a laparoscope into joints, abdominal organs, the bladder wall and body cavities.

Modified Host Cells

The present disclosure provides a modified cell comprising a Cas12L polypeptide of the present disclosure and/or a nucleic acid comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure. The present disclosure provides a modified cell comprising a Cas12L polypeptide of the present disclosure, where the modified cell is a cell that does not normally comprise a Cas12L polypeptide of the present disclosure. The present disclosure provides a modified cell (e.g., a genetically modified cell) comprising nucleic acid comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with an mRNA comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising: a) a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure; and b) a nucleotide sequence encoding a Cas12L guide RNA of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising: a) a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure; b) a nucleotide sequence encoding a Cas12L guide RNA of the present disclosure; and c) a nucleotide sequence encoding a donor template.

A cell that serves as a recipient for a Cas12L polypeptide of the present disclosure and/or a nucleic acid comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure and/or a Cas12L guide RNA of the present disclosure, can be any of a variety of cells, including, e.g., in vitro cells; in vivo cells; ex vivo cells; primary cells; cancer cells; animal cells; plant cells; algal cells; fungal cells; etc. A cell that serves as a recipient for a Cas12L polypeptide of the present disclosure and/or a nucleic acid comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure and/or a Cas12L guide RNA of the present disclosure is referred to as a “host cell” or a “target cell.” A host cell or a target cell can be a recipient of a Cas12L system of the present disclosure. A host cell or a target cell can be a recipient of a Cas12L RNP of the present disclosure. A host cell or a target cell can be a recipient of a single component of a Cas12L system of the present disclosure.

Non-limiting examples of cells (target cells) include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatos, rice, cassava, sugarcane, pumpkin, hay, potatos, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like. In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).

A cell can be an in vitro cell (e.g., established cultured cell line). A cell can be an ex vivo cell (cultured cell from an individual). A cell can be and in vivo cell (e.g., a cell in an individual). A cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be an organism. A cell can be a cell in a cell culture (e.g., in vitro cell culture). A cell can be one of a collection of cells. A cell can be a prokaryotic cell or derived from a prokaryotic cell. A cell can be a bacterial cell or can be derived from a bacterial cell. A cell can be an archaeal cell or derived from an archaeal cell. A cell can be a eukaryotic cell or derived from a eukaryotic cell. A cell can be a plant cell or derived from a plant cell. A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. A cell can be a microbe cell or derived from a microbe cell. A cell can be a fungi cell or derived from a fungi cell. A cell can be an insect cell. A cell can be an arthropod cell. A cell can be a protozoan cell. A cell can be a helminth cell.

Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc.

Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autotransplated expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells.

In some cases, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells.

Adult stem cells are resident in differentiated tissue, but retain the properties of self-renewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.

Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell is a human stem cell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a non-human primate stem cell.

Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4, CDH17, and PPARGC1A.

In some embodiments, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34+ and CD3⁻. HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

In other embodiments, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art.

In other embodiments, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC.

A cell is in some cases a plant cell. A plant cell can be a cell of a monocotyledon. A cell can be a cell of a dicotyledon.

In some cases, the cell is a plant cell. For example, the cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes, Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like. As another example, the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, chinese artichoke (crosnes), chinese cabbage, chinese celery, chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, corn stalks, corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive, escarole, fiddle head ferns, field cress, frisee, gai choy (chinese mustard), gailon, galanga (siam, thai ginger), garlic, ginger root, gobo, greens, hanover salad greens, huauzontle, jerusalem artichokes, jicama, kale greens, kohlrabi, lamb's quarters (quilete), lettuce (bibb), lettuce (boston), lettuce (boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lolla rossa), lettuce (oak leaf—green), lettuce (oak leaf—red), lettuce (processed), lettuce (red leaf), lettuce (romaine), lettuce (ruby romaine), lettuce (russian red mustard), linkok, lo bok, long beans, lotus root, mache, maguey (agave) leaves, malanga, mesculin mix, mizuna, moap (smooth luffa), moo, moqua (fuzzy squash), mushrooms, mustard, nagaimo, okra, ong choy, onions green, opo (long squash), ornamental corn, ornamental gourds, parsley, parsnips, peas, peppers (bell type), peppers, pumpkins, radicchio, radish sprouts, radishes, rape greens, rape greens, rhubarb, romaine (baby red), rutabagas, salicornia (sea bean), sinqua (angled/ridged luffa), spinach, squash, straw bales, sugarcane, sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taro shoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes, tomatoes (cherry), tomatoes (grape type), tomatoes (plum type), tumeric, turnip tops greens, turnips, water chestnuts, yampi, yams (names), yu choy, yuca (cassava), and the like.

In some cases, the plant cell is a cell of a plant component such as a leaf, a stem, a root, a seed, a flower, pollen, an anther, an ovule, a pedicel, a fruit, a meristem, a cotyledon, a hypocotyl, a pod, an embryo, endosperm, an explant, a callus, or a shoot.

A cell is in some cases an arthropod cell. For example, the cell can be a cell of a sub-order, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera, Embioptera, Orthoptera, Zoraptera, Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea, Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Holometabola, Hymenoptera, Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera, Mecoptera, Siphonaptera, Diptera, Trichoptera, or Lepidoptera.

A cell is in some cases an insect cell. For example, in some cases, the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.

Kits

The present disclosure provides a kit comprising a Cas12L system of the present disclosure, or a component of a Cas12L system of the present disclosure.

A kit of the present disclosure can comprise: a) a Cas12L polypeptide of the present disclosure and a Cas12L guide RNA; b) a Cas12L polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; c) a Cas12L fusion polypeptide of the present disclosure and a Cas12L guide RNA; d) a Cas12L fusion polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; e) an mRNA encoding a Cas12L polypeptide of the present disclosure; and a Cas12L guide RNA; f) an mRNA encoding a Cas12L polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; g) an mRNA encoding a Cas12L fusion polypeptide of the present disclosure; and a Cas12L guide RNA; h) an mRNA encoding a Cas12L fusion polypeptide of the present disclosure, a Cas12L guide RNA, and a donor template nucleic acid; i) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure and a nucleotide sequence encoding a Cas12L guide RNA; j) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, a nucleotide sequence encoding a Cas12L guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; k) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure and a nucleotide sequence encoding a Cas12L guide RNA; 1) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, a nucleotide sequence encoding a Cas12L guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; m) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; n) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; and a donor template nucleic acid; o) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; p) a first recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12L guide RNA; and a donor template nucleic acid; q) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure, a nucleotide sequence encoding a first Cas12L guide RNA, and a nucleotide sequence encoding a second Cas12L guide RNA; or r) a recombinant expression vector comprising a nucleotide sequence encoding a Cas12L fusion polypeptide of the present disclosure, a nucleotide sequence encoding a first Cas12L guide RNA, and a nucleotide sequence encoding a second Cas12L guide RNA; or some variation of one of (a) through (r).

A kit of the present disclosure can comprise: a) a component, as described above, of a Cas12L system of the present disclosure, or can comprise a Cas12L system of the present disclosure; and b) one or more additional reagents, e.g., i) a buffer; ii) a protease inhibitor; iii) a nuclease inhibitor; iv) a reagent required to develop or visualize a detectable label; v) a positive and/or negative control target DNA; vi) a positive and/or negative control Cas12L guide RNA; and the like. A kit of the present disclosure can comprise: a) a component, as described above, of a Cas12L system of the present disclosure, or can comprise a Cas12L system of the present disclosure; and b) a therapeutic agent.

A kit of the present disclosure can comprise a recombinant expression vector comprising: a) an insertion site for inserting a nucleic acid comprising a nucleotide sequence encoding a portion of a Cas12L guide RNA that hybridizes to a target nucleotide sequence in a target nucleic acid; and b) a nucleotide sequence encoding the Cas12L-binding portion of a Cas12L guide RNA. A kit of the present disclosure can comprise a recombinant expression vector comprising: a) an insertion site for inserting a nucleic acid comprising a nucleotide sequence encoding a portion of a Cas12L guide RNA that hybridizes to a target nucleotide sequence in a target nucleic acid; b) a nucleotide sequence encoding the Cas12L-binding portion of a Cas12L guide RNA; and c) a nucleotide sequence encoding a Cas12L polypeptide of the present disclosure.

Utility

A Cas12L polypeptide of the present disclosure, or a Cas12L fusion polypeptide of the present disclosure, finds use in a variety of methods (e.g., in combination with a Cas12L guide RNA and in some cases further in combination with a donor template). For example, a Cas12L polypeptide of the present disclosure can be used to (i) modify (e.g., cleave, e.g., nick; methylate; etc.) target nucleic acid (DNA or RNA; single stranded or double stranded); (ii) modulate transcription of a target nucleic acid; (iii) label a target nucleic acid; (iv) bind a target nucleic acid (e.g., for purposes of isolation, labeling, imaging, tracking, etc.); (v) modify a polypeptide (e.g., a histone) associated with a target nucleic acid; and the like. Thus, the present disclosure provides a method of modifying a target nucleic acid. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with: a) a Cas12L polypeptide of the present disclosure; and b) one or more (e.g., two) Cas12L guide RNAs. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with: a) a Cas12L polypeptide of the present disclosure; b) a Cas12L guide RNA; and c) a donor nucleic acid (e.g, a donor template). In some cases, the contacting step is carried out in a cell in vitro. In some cases, the contacting step is carried out in a cell in vivo. In some cases, the contacting step is carried out in a cell ex vivo.

Because a method that uses a Cas12L polypeptide includes binding of the Cas12L polypeptide to a particular region in a target nucleic acid (by virtue of being targeted there by an associated Cas12L guide RNA), the methods are generally referred to herein as methods of binding (e.g., a method of binding a target nucleic acid). However, it is to be understood that in some cases, while a method of binding may result in nothing more than binding of the target nucleic acid, in other cases, the method can have different final results (e.g., the method can result in modification of the target nucleic acid, e.g., cleavage/methylation/etc., modulation of transcription from the target nucleic acid; modulation of translation of the target nucleic acid; genome editing; modulation of a protein associated with the target nucleic acid; isolation of the target nucleic acid; etc.).

For examples of suitable methods, see, for example, Jinek et al., Science. 2012 Aug. 17;337(6096):816-21; Chylinski et al., RNA Biol. 2013 May; 10(5):726-37; Ma et al., Biomed Res Int. 2013; 2013:270805; Hou et al., Proc Natl Acad Sci USA. 2013 Sep. 24;110(39):15644-9; Jinek et al., Elife. 2013;2:e00471; Pattanayak et al., Nat Biotechnol. 2013 September;31(9):839-43; Qi et al, Cell. 2013 Feb. 28;152(5):1173-83; Wang et al., Cell. 2013 May 9;153(4):910-8; Auer et al., Genome Res. 2013 Oct. 31; Chen et al., Nucleic Acids Res. 2013 Nov. 1;41(20):e19; Cheng et al., Cell Res. 2013 October;23(10):1163-71; Cho et al., Genetics. 2013 November; 195(3):1177-80; DiCarlo et al., Nucleic Acids Res. 2013 April; 41(7):4336-43; Dickinson et al., Nat Methods. 2013 Oct;10(10):1028-34; Ebina et al., Sci Rep. 2013; 3:2510; Fujii et al, Nucleic Acids Res. 2013 Nov. 1;41(20):e187; Hu et al., Cell Res. 2013 November; 23(11):1322-5; Jiang et al., Nucleic Acids Res. 2013 Nov. 1;41(20):e188; Larson et al., Nat Protoc. 2013 November; 8(11):2180-96; Mali et. at., Nat Methods. 2013 October;10(10):957-63; Nakayama et al., Genesis. 2013 December; 51(12):835-43; Ran et al., Nat Protoc. 2013 November; 8(11):2281-308; Ran et al., Cell. 2013 Sep. 12;154(6):1380-9; Upadhyay et al., G3 (Bethesda). 2013 Dec. 9;3(12):2233-8; Walsh et al., Proc Natl Acad Sci U.S. A. 2013 Sep. 24;110(39):15514-5; Xie et al., Mol Plant. 2013 Oct. 9; Yang et al., Cell. 2013 Sep. 12;154(6):1370-9; and U.S. patents and patent applications: U.S. Pat. Nos. 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919; 20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; each of which is hereby incorporated by reference in its entirety.

For example, the present disclosure provides (but is not limited to) methods of cleaving a target nucleic acid; methods of editing a target nucleic acid; methods of modulating transcription from a target nucleic acid; methods of isolating a target nucleic acid, methods of binding a target nucleic acid, methods of imaging a target nucleic acid, methods of modifying a target nucleic acid, and the like.

As used herein, the terms/phrases “contact a target nucleic acid” and “contacting a target nucleic acid”, for example, with a Cas12L polypeptide or with a Cas12L fusion polypeptide, etc., encompass all methods for contacting the target nucleic acid. For example, a Cas12L polypeptide can be provided to a cell as protein, RNA (encoding the Cas12L polypeptide), or DNA (encoding the Cas12L polypeptide); while a Cas12L guide RNA can be provided as a guide RNA or as a nucleic acid encoding the guide RNA. As such, when, for example, performing a method in a cell (e.g., inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo), a method that includes contacting the target nucleic acid encompasses the introduction into the cell of any or all of the components in their active/final state (e.g., in the form of a protein(s) for Cas12L polypeptide; in the form of a protein for a Cas12L fusion polypeptide; in the form of an RNA in some cases for the guide RNA), and also encompasses the introduction into the cell of one or more nucleic acids encoding one or more of the components (e.g., nucleic acid(s) comprising nucleotide sequence(s) encoding a Cas12L polypeptide or a Cas12L fusion polypeptide, nucleic acid(s) comprising nucleotide sequence(s) encoding guide RNA(s), nucleic acid comprising a nucleotide sequence encoding a donor template, and the like). Because the methods can also be performed in vitro outside of a cell, a method that includes contacting a target nucleic acid, (unless otherwise specified) encompasses contacting outside of a cell in vitro, inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo, etc.

In some cases, a method of the present disclosure for modifying a target nucleic acid comprises introducing into a target cell a Cas12L locus, e.g., a nucleic acid comprising a nucleotide sequence encoding a Cas12L polypeptide as well as nucleotide sequences of about 1 kilobase (kb) to 5 kb in length surrounding the Cas12L-encoding nucleotide sequence from a cell (e.g., in some cases a cell that in its natural state (the state in which it occurs in nature) comprises a Cas12L locus) comprising a Cas12L locus, where the target cell does not normally (in its natural state) comprise a Cas12L locus. However, one or more spacer sequences, encoding guide sequences for the encoded crRNA(s), can be modified such that one or more target sequences of interest are targeted. Thus, for example, in some cases, a method of the present disclosure for modifying a target nucleic acid comprises introducing into a target cell a Cas12L locus, e.g., a nucleic acid obtained from a source cell (e.g., in some cases a cell that in its natural state (the state in which it occurs in nature) comprises a Cas12L locus), where the nucleic acid has a length of from 100 nucleotides (nt) to 5 kb in length (e.g., from 100 nt to 500 nt, from 500 nt to 1 kb, from 1 kb to 1.5 kb, from 1.5 kb to 2 kb, from 2 kb to 2.5 kb, from 2.5 kb to 3 kb, from 3 kb to 3.5 kb, from 3.5 kb to 4 kb, or from 4 kb to 5 kb in length) and comprises a nucleotide sequence encoding a Cas12L polypeptide. As noted above, in some such cases, one or more spacer sequences, encoding guide sequences for the encoded crRNA(s), can be modified such that one or more target sequences of interest are targeted. In some cases, the method comprises introducing into a target cell: i) a Cas12L locus; and ii) a donor DNA template. In some cases, the target nucleic acid is in a cell-free composition in vitro. In some cases, the target nucleic acid is present in a target cell. In some cases, the target nucleic acid is present in a target cell, where the target cell is a prokaryotic cell. In some cases, the target nucleic acid is present in a target cell, where the target cell is a eukaryotic cell. In some cases, the target nucleic acid is present in a target cell, where the target cell is a mammalian cell. In some cases, the target nucleic acid is present in a target cell, where the target cell is a plant cell.

In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a Cas12L polypeptide of the present disclosure, or with a Cas12L fusion polypeptide of the present disclosure. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a Cas12L polypeptide and a Cas12L guide RNA. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a Cas12L polypeptide, a first Cas12L guide RNA, and a second Cas12L guide RNA In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a Cas12L polypeptide of the present disclosure and a Cas12L guide RNA and a donor DNA template.

Target Nucleic Acids and Target Cells of Interest

A Cas12L polypeptide of the present disclosure, or a Cas12L fusion polypeptide of the present disclosure, when bound to a Cas12L guide RNA, can bind to a target nucleic acid, and in some cases, can bind to and modify a target nucleic acid. A target nucleic acid can be any nucleic acid (e.g., DNA, RNA), can be double stranded or single stranded, can be any type of nucleic acid (e.g., a chromosome (genomic DNA), derived from a chromosome, chromosomal DNA, plasmid, viral, extracellular, intracellular, mitochondrial, chloroplast, linear, circular, etc.) and can be from any organism (e.g., as long as the Cas12L guide RNA comprises a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid, such that the target nucleic acid can be targeted).

A target nucleic acid can be DNA or RNA. A target nucleic acid can be double stranded (e.g., dsDNA, dsRNA) or single stranded (e.g., ssRNA, ssDNA). In some cases, a target nucleic acid is single stranded. In some cases, a target nucleic acid is a single stranded RNA (ssRNA). In some cases, a target ssRNA (e.g., a target cell ssRNA, a viral ssRNA, etc.) is selected from: mRNA, rRNA, tRNA, non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and microRNA (miRNA). In some cases, a target nucleic acid is a single stranded DNA (ssDNA) (e.g., a viral DNA). As noted above, in some cases, a target nucleic acid is single stranded.

A target nucleic acid can be located anywhere, for example, outside of a cell in vitro, inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo. Suitable target cells (which can comprise target nucleic acids such as genomic DNA) include, but are not limited to: a bacterial cell; an archaeal cell; a cell of a single-cell eukaryotic organism; a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); an animal cell; a cell from an invertebrate animal (e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.); a cell of an insect (e.g., a mosquito; a bee; an agricultural pest; etc.); a cell of an arachnid (e.g., a spider; a tick; etc.); a cell from a vertebrate animal (e.g., a fish, an amphibian, a reptile, a bird, a mammal); a cell from a mammal (e.g., a cell from a rodent; a cell from a human; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an ungulate (e.g., a cow, a horse, a camel, a llama, a vicuna, a sheep, a goat, etc.); a cell of a marine mammal (e.g., a whale, a seal, an elephant seal, a dolphin, a sea lion; etc.) and the like. Any type of cell may be of interest (e.g. a stem cell, e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.).

Cells may be from established cell lines or they may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture. For example, primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Typically, the primary cell lines are maintained for fewer than 10 passages in vitro. Target cells can be unicellular organisms and/or can be grown in culture. If the cells are primary cells, they may be harvest from an individual by any convenient method. For example, leukocytes may be conveniently harvested by apheresis, leukocytapheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be conveniently harvested by biopsy.

In some of the above applications, the subject methods may be employed to induce target nucleic acid cleavage, target nucleic acid modification, and/or to bind target nucleic acids (e.g., for visualization, for collecting and/or analyzing, etc.) in mitotic or post-mitotic cells in vivo and/or ex vivo and/or in vitro (e.g., to disrupt production of a protein encoded by a targeted mRNA, to cleave or otherwise modify target DNA, to geneically modify a target cell, and the like). Because the guide RNA provides specificity by hybridizing to target nucleic acid, a mitotic and/or post-mitotic cell of interest in the disclosed methods may include a cell from any organism (e.g. a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like, a fungal cell (e.g., a yeast cell), an animal cell, a cell from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal, a cell from a rodent, a cell from a human, etc.). In some cases, a subject Cas12L protein (and/or nucleic acid encoding the protein such as DNA and/or RNA), and/or Cas12L guide RNA (and/or a DNA encoding the guide RNA), and/or donor template, and/or RNP can be introduced into an individual (i.e., the target cell can be in vivo) (e.g., a mammal, a rat, a mouse, a pig, a primate, a non-human primate, a human, etc.). In some case, such an administration can be for the purpose of treating and/or preventing a disease, e.g., by editing the genome of targeted cells.

Plant cells include cells of a monocotyledon, and cells of a dicotyledon. The cells can be root cells, leaf cells, cells of the xylem, cells of the phloem, cells of the cambium, apical meristem cells, parenchyma cells, collenchyma cells, sclerenchyma cells, and the like. Plant cells include cells of agricultural crops such as wheat, corn, rice, sorghum, millet, soybean, etc. Plant cells include cells of agricultural fruit and nut plants, e.g., plant that produce apricots, oranges, lemons, apples, plums, pears, almonds, etc.

Additional examples of target cells are listed above in the section titled “Modified cells.” Non-limiting examples of cells (target cells) include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatos, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like. In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).