As such, a gene drive is capable of self-propagating through a population by a mechanism in which the target-site directed nuclease cuts a target DNA sequence at a specific site in a target locus that does not encode the drive, inducing the cell to repair the damage by copying the drive sequence onto the damaged chromosome. The cell then has two copies of the drive sequence. As such a gene drive—and in particular an exogenous nucleotide sequence—is stably introduced in to the germline cell of a desired organism.

A resulting transgenic organism may be developed from the germline cell. The resulting transgenic organism can then be introduced into a wild type population, and through mating by the transgenic organism with a wild type organism, the exogenous nucleic acid sequence is transferred to resulting offspring or progeny. The methods of the present invention are thus directed to the production of a population of transgenic organisms having desired traits from one of a plurality of initial transgenic organisms and a wild type organism.

The gene drive of the present invention may comprise an exogenous nucleotide sequence located between two flanking sequences.

The exogenous nucleotide sequence comprises, or encodes, a first entity which is a component of a homing nuclease.

Suitably, the component of the homing nuclease may be a targeting entity which is capable of targeting a nuclease to a target sequence and/or a nuclease.

The entity which is capable of a targeting a nuclease to a target sequence may be a polynucleotide which is capable of directing a nuclease to a target site, a polynucleotide encoding a polypeptide which is capable of directing a nuclease to a target site or a polynucleotide which encodes a target-specific nuclease.

The exogenous nucleotide sequence may comprise a nucleic acid sequence which is, or encodes, a nuclease targeting entity. The exogenous nucleotide sequence may comprise a nucleic acid sequence which encodes a nuclease.

The exogenous nucleotide sequence may comprise a nucleic acid sequence which is, or encodes, a nuclease targeting entity and a nucleic acid sequence which encodes a nuclease.

The exogenous nucleotide sequence also may comprise at least one promoter such that the component of the homing nuclease can be expressed. The particular promoter will depend on the target organism. The promoter may be a constitutive promoter or an inducible promoter. The choice of a suitable promoter is routine in the art. The exogenous nucleotide sequence may further comprise any other nucleic acid sequences required for expression of the nucleic acid sequences provided by the exogenous nucleotide sequence, in particular in a germline cell.

The exogenous nucleotide sequence may also comprise any further nucleic acid sequences which it is desired to be expressed in the cell, in particular in the germline cell. Such further nucleic acid sequences may be referred to as “cargo nucleic acid”. It is within the ambit of the skilled person to provide suitable cargo nucleic acid to achieve the desired trait. For example, the cargo nucleic acid may comprise a polypeptide-encoding sequence which is to be introduced into a population. Alternatively, the cargo nucleic acid may comprise a mutant copy of the corresponding wild type sequence present in the genome of the target population. The mutant may, for example, comprise a deletion, addition or substitution compared to the corresponding wild type sequence.

The exogenous nucleotide sequence may be inserted into a gene in the target genome, such that the function or activity of that gene is reduced, disrupted or deleted.

As described herein, in the “overwriting” embodiment the exogenous nucleotide sequence may also comprise a target site for each of the other gene drives of the multiplex gene drive system. For example, if the method comprises introducing a first gene drive into the germline genomic DNA of a first subpopulation and introducing a second gene drive into the germline genomic DNA of a second subpopulation; the exogenous nucleotide sequence of the first gene drive may comprise a target site for the nuclease targeting entity of the second gene drive and the exogenous nucleotide sequence of the second gene drive may comprise a target site for the nuclease targeting entity of the first gene drive.

If the method comprises introducing a first gene drive into the germline genomic DNA of a first subpopulation; a second gene drive into the germline genomic DNA of a second subpopulation, and a third gene drive into the germline genomic DNA of a third subpopulation; the exogenous nucleotide sequence of the first gene drive may comprise a target site for the nuclease targeting entity of the second gene drive and the nuclease targeting entity of the third gene drive; the exogenous nucleotide sequence of the second gene drive may comprise a target site for the nuclease targeting entity of the first gene drive and the nuclease targeting entity of the third gene drive; and the exogenous nucleotide sequence of the third gene drive may comprise a target site for the nuclease targeting entity of the first gene drive and the nuclease targeting entity of the second gene drive.

Each further gene drive introduced into a further subpopulation, for example a fourth, fifth or sixth gene drive may comprise an exogenous nucleotide sequence which comprises a different nuclease targeting entity and a target site for the nuclease targeting entity of each of the other gene drive of the multiplex system.

As described herein, in the “blocking” embodiment the exogenous nucleotide sequence may not comprise a target site for one or more, in particular each, of the other gene drives of the multiplex gene drive system. For example, if the method comprises introducing a first gene drive into the germline genomic DNA of a first subpopulation and introducing a second gene drive into the germline genomic DNA of a second subpopulation; the exogenous nucleotide sequence of the first gene drive does not comprise a target site for the nuclease targeting entity of the second gene drive and the exogenous nucleotide sequence of the second gene drive does not comprise a target site for the nuclease targeting entity of the first gene drive.

If the method comprises introducing a first gene drive into the germline genomic DNA of a first subpopulation; a second gene drive into the germline genomic DNA of a second subpopulation, and a third gene drive into the germline genomic DNA of a third subpopulation; the exogenous nucleotide sequence of the first gene drive does not comprise a target site for the nuclease targeting entity of the second gene drive or the nuclease targeting entity of the third gene drive; the exogenous nucleotide sequence of the second gene drive does not comprise a target site for the nuclease targeting entity of the first gene drive or the nuclease targeting entity of the third gene drive; and the exogenous nucleotide sequence of the third gene drive does not comprise a target site for the nuclease targeting entity of the first gene drive or the nuclease targeting entity of the second gene drive.

In the “blocking” embodiment, each further gene drive introduced into a further subpopulation, for example a fourth, fifth or sixth gene drive may comprise an exogenous nucleotide sequence which comprises a different nuclease targeting entity; but the exogenous nucleotide sequence does not comprise a target site for the nuclease targeting entity of each of the other gene drive of the multiplex system.

As used herein, an exogenous nucleotide sequence means a sequence which differs from the corresponding sequence of the wild-type genome. The exogenous sequence may differ from the wild-type sequence by at least 1, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 2000 or at least 10000 nucleotides.

As described herein, the exogenous nucleotide sequence may include an entity which is capable of targeting a nuclease to a target site.

Exogenous polynucleotides, such as the gene drive constructs of the present invention, can be introduced into a cell using any method in the art which is suitable for such introduction. Suitable methods include, for example, transfection, transduction, transformation, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment and conjugation.

Engineered Subpopulation

The present invention relates to generating a plurality of engineered subpopulations, wherein each engineered subpopulation comprises a separate gene drive of the present invention.

Suitably, a plurality may be 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more.

The invention may comprise preparing a plurality of engineered subpopulations as described herein and introducing them into a general population (e.g. a wild type population).

The engineered subpopulation, and general population to be targeted includes, but is not limited to, insects, mammals, plants, and fungi.

Suitably, the term mammal is used to refer to a non-human mammal.

Germline Genomic DNA

Germline cells of the present disclosure include any germline cell into which a gene drive is to be introduced and expressed as described herein. Suitable germline cells include, but are not limited to, eukaryotic germline cells, prokaryotic germline cells, insect germline cells, mammalian germline cells, plant germline cells, fungal germline cells.

The germline cell may be homozygous or heterozygous for the gene drive construct comprising the exogenous nucleotide sequence. Suitably, the germline cell may be homozygous for the gene drive construct, for example when the target locus is a non-coding DNA sequence or a putatively non-functional locus; is not a fertility- or viability-essential locus; and/or is a fitness-neutral locus.

Suitably, the gene drive construct may be introduced into a zygote, a gamete or a cell that can give rise to a gamete.

Nuclease Targeting Entity/Nuclease

The nuclease used herein may be any suitable homing nuclease which requires a long recognition sequence for productive cleavage. In other words the nuclease preferably only cleaves at one site in the genome of the target population.

The nuclease targeting entity targets the nuclease to a single target site in the target locus. Suitably, the nuclease targeting entity does not target the nuclease to more than one target site in the target locus.

Suitable nucleases (or nuclease systems) include, but are not limited to, RNA-guided nucleases (e.g. CRISPR-nuclease), homing endonucleases, TALENs and zinc-finger nucleases.

The nuclease may induce double-strand DNA breaks or single-strand DNA breaks (also referred to as a nickase).

The nuclease activity (e.g. the nuclease enzyme) or the nuclease targeting entity may not be encoded by the gene drive construct and may be encoded by a separate polynucleotide.

Suitably, the exogenous sequence of the gene drive construct may comprise a polynucleotide sequence which is, or encodes, a nuclease targeting entity.

Suitably, the exogenous sequence of the gene drive construct may comprise a polynucleotide sequence which encodes the nuclease.

Preferably the exogenous sequence of the gene drive construct comprises a polynucleotide sequence which is, or encodes, a nuclease targeting entity and a polynucleotide sequence which encodes the nuclease.

The nuclease targeting entity may be any entity which confers target specificity on the nuclease. The nuclease targeting entity may be a polynucleotide which is capable of directing a nuclease to a target site, a polynucleotide encoding a polypeptide which is capable of directing a nuclease to a target site or a polynucleotide which encodes a target-specific nuclease.

In embodiments in which the nuclease is a RNA-guided nuclease (e.g. CRISPR-nuclease), the nuclease targeting entity may be any RNA which confers target specificity on the nuclease. For example, the nuclease targeting entity may be a CRISPR-RNA (crRNA) or a single-guide RNA (sgRNA). The RNA may be between about 100 to about 500 nucleotides. The RNA may be between about 20 to about 100, or about 20 to about 50 nucleotides.

Suitable RNA-guided nucleases are known in the art and include Type II CRISPR nucleases.

Suitable RNA-guided nucleases include, for example, Cas9, Cpf1 or Csm1 nucleases.

Suitably, the exogenous nucleotide sequence may comprise nucleic acid sequences which between them encode all the elements necessary to provide a functional RNA-guided nuclease which cleaves at the target site of the nuclease targeting entity (i.e. the crRNA or sgRNA). For example, the exogenous nucleotide sequence may comprise a nucleic acid sequence encoding a gRNA or a crRNA and a nucleic acid sequence encoding an RNA-guided nuclease. The exogenous nucleotide sequence may comprise a nucleic acid sequence encoding a gRNA and a nucleic acid sequence encoding an RNA-guided nuclease.

Suitably, the exogenous nucleotide sequence may comprise a nucleic acid sequence encoding a homing endonuclease, TALEN or zinc-finger nuclease.

Target Sequence

“Target sequence”, as used herein, may be synonymous with the term “target site”.

The target sequence/target site may be any nucleic acid sequence to which a nuclease targeting entity as described herein directs a nuclease to either cut or nick the genomic DNA. In the context of the present disclosure, each target site is located within the target locus of the genomic DNA of the target population.

The target site may be located within the target locus of the wild-type genomic DNA of the target population. The target site may be located with a target locus associated with a resistance allele; for example an insecticide-resistance allele or within the exogenous nucleotide sequence of another gene drive construct.

The target site may be present within a gene drive construct as described herein.

The target site may refer to the nucleic acid sequence to which the nuclease targeting entity and nuclease bind or the nucleic acid sequence at which the nuclease cleaves or nicks following targeting by the nuclease targeting entity.

By way of example, the target site of a CRISPR-nuclease is typically about 20 nucleotides in length and is selected based on at least the following criteria: (i) the sequence is unique compared to the rest of the genome; and (ii) the target site is adjacent to a Protospacer Adjacent Motif (PAM) sequence.

Approaches and techniques for selecting appropriate target sites are known in the art. Suitably PAM sequences for different CRISPR-nucleases are described in Karvelis et al. (Methods. 2017 May 15; 121-122:3-8) (hereby incorporated by reference).

Suitably, the target site of each nuclease targeting entity used in the present method should be unique and non-overlapping. In other words, the nuclease targeting entity of each gene drive should target a nuclease to cut or nick at a different site in the target genome such that there is no direct interaction between the nuclease activities at each target site.

Thus, each target site is preselected to be independent of the target sites for each of the other nuclease targeting entities provided by other gene drive constructs used in the present method.

Accordingly, the distance between target sites may depend on the nuclease targeting entity and nuclease to be used. Target site requirements for suitably nuclease targeting entities and corresponding nucleases are well known in the art.

Suitably, each target site may be less than 0.5 kb, less than 1 kb, less than 2 kb, less than 5 kb, or less than 10 kb apart in the genome.

Flanking Sequence

Each gene drive construct also comprises at least two flanking sequences, one positioned on each side of the exogenous nucleotide sequence. Thus in each gene drive construct flanking sequences are positioned on opposite sides of the exogenous nucleotide sequence, such that the exogenous nucleotide sequence is between the flanking sequences. Suitably, the flanking sequences include at least a sequence which is essentially identical to a corresponding sequence in a selected target locus of the target organism.

Such flanking sequences allow a cell to insert the exogenous nucleotide sequence into its genome DNA at a cut site using well-understood mechanisms such as homologous recombination.

The flanking sequence may be any length which is suitable to allow a cell to insert the exogenous nucleotide sequence into its genome DNA at a cut site through homologous recombination or another homology-directed repair process.

Suitably, the flanking sequence may be 50 to 2000 nucleotides, 100 to 1500 nucleotides, 100 to 1000 nucleotides, 100 to 500 nucleotides or 100 to 250 nucleotides in length.

Suitably, the flanking sequences of each gene drive construct may comprise essentially identical sequences to the other gene drive constructs. In other words, the first and second flanking sequences of a first gene drive construct may comprise essentially identical sequences to the third and fourth flanking sequences of a second gene drive construct; respectively. Suitably, the flanking sequences of each gene drive construct may comprise different sequences. In other words, the first and second flanking sequences of a first gene drive construct may comprise different sequences to the third and fourth flanking sequences of a second gene drive construct; respectively.

By way of example, in the overwriting strategy of the present invention, the flanking sequences of each gene drive construct may comprise essentially identical sequences such that each pair of flanking sequences is essentially identical to the same corresponding sequence in a selected target locus. As such, successful homing by any gene drive construct will remove some or all endogenous target sites in the target locus and introduce target sites for each of the nuclease targeting entities present in the other gene drive constructs into to the target locus.

Target Locus

“Target locus” is used herein to refer to a genomic region into which the gene drive construct is to be inserted.

The target locus comprises a target site for the nuclease targeting entity, which target site is located between nucleic acid sequences which are essentially identical to the flanking sequences of the gene drive construct (such that the cell can insert the exogenous nucleotide sequence into its genome DNA at a cut site through homologous recombination or another homology-directed repair process).

The target locus may be at least 0.5 kb, at least 1 kb, at least 2 kb, at least 5 kb or at least 10 kb in length.

The target locus may be a gene or a non-coding sequence or putatively non-functional locus.

The gene may be a protein-coding gene or a gene encoding a functional RNA (e.g. lncRNA or miRNA). The target locus may be in an exon or an intron.

The methods of the present invention are particularly advantageous for targeting a putatively non-functional locus, or locus which is not fertility- or viability-essential. These loci would not be expected to confer a fitness advantage on the individual. As such, drive-resistant deletion mutants will likely retain full fitness and would be expected to propagate throughout a population leading to loss of the gene drive.

Suitably, the target locus may be a putatively non-functional locus; a locus which is not essential for fertility or a locus which is not essential for viability.

As used herein, fitness-essential or viability-essential means that reduced function or loss of function of a gene results in a substantially lethal or sterile phenotype.

Suitably, the target locus may be a fitness-neutral locus.

Suitably, the target locus is not located on the X or Y chromosome.

Vector

The present invention also provides a vector, or kit of vectors which comprises a gene drive construct of the invention. Such a vector may be used to introduce the nucleic acid sequence(s) or construct(s) into a host cell.

The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.

The vector may be capable of transfecting or transducing a cell.

Kit

The present invention also provides a plurality of gene drive constructs of the present invention or a kit comprising a plurality of gene drive constructs of the present invention.

Use

The present invention further provides the use of a first and a second nucleic acid gene drive construct of the invention to introduce a first and a second gene drive into a population.

The nucleic acid gene drive construct is a gene drive construct as defined herein. The gene drive construct comprises an exogenous nucleotide sequence as defined herein.

The invention further provides the use of a third nucleic acid gene drive construct of the invention to introduce a third gene drive into the population.

The invention further provides the use of a fourth, a fifth or a sixth nucleic acid gene drive construct of the invention to introduce a fourth, fifth or sixth gene drive into the population, wherein the fourth, a fifth or a sixth nucleic acid construct is a nucleic acid construct as defined herein.

As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.

It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Suitably, the polynucleotides of the present invention are codon optimised to enable expression in a mammalian cell or insect cell, in particular a germline cell.

Nucleic acids according to the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3′ and/or 5′ ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.