Gene constructs for population inhibition

Abstract

The present invention relates to gene constructs, in particular to gene constructs for population inhibition and pest control. In particular, the present invention relates to a gene construct capable of disrupting a haploid sufficient gene required for the survival or reproduction of an organism wherein the gene construct encodes a genomic editor that produces dominant lethal or sterile mutations in a target gene of the organism. The invention also relates to methods of inhibiting wild-type populations by using the gene constructs described herein.

Description

Technical Field

[0001] The present invention relates to genetic constructs, and more particularly to genetic constructs for population suppression and pest control. Specifically, the present invention relates to genetic constructs capable of disrupting a haplosufficient gene required for survival or reproduction of an organism, wherein the genetic construct encodes a genome editor that generates a dominant lethal or sterility mutation in a target gene of the organism. The present invention also relates to methods for suppressing wild-type populations using the genetic constructs described herein. Background Art

[0002] Currently, pest control is primarily based on the use of chemical agents. Other alternatives include physical control (including nets or fences) and biological control (e.g., introduced predators). Genetic biological control strategies may have advantages due to their species specificity, eco-friendliness, and ability to exploit a species' natural courtship behavior for spread. Several genetic control methods exist. The most widely used is the sterile insect technique (SIT). A successful example is the control of the New World screwworm, currently being implemented through programs such as the Panama Screwworm Isolation Zone Maintenance Program, jointly managed by the governments of the United States and Panama. Other applications include the control of the Mediterranean fruit fly (an agricultural pest) implemented by government agencies such as the FOA and the IAEA. Releasing insects carrying a dominant lethal gene (RIDL) is another alternative, developed and commercialized by the private company Oxitec. Releasing mosquitoes carrying the Wolbachia bacterium has been developed by companies such as Verily and Mosquito Mate for population suppression, or by non-profit organizations such as the World Mosquito Project for population modification.

[0003] As mentioned above, SIT has been used to control certain pest populations, but for other pest populations, it is not effective enough to provide satisfactory control effects at a reasonable price. There are currently many proposals for more efficient localized genetic biocontrol. The Y-linked editor (YLE) and fs-RIDL-Drive constructs modeled by Burt and Deredec (2018, P. Roy. Soc. Biol. Sci. 285 (1883)) are expected to provide effective and localized control, but they have some shortcomings that make them difficult to use in certain species. YLE needs to be expressed from the Y chromosome, which may be difficult to achieve and also means that they are not suitable for species without Y chromosomes. In addition, fs-RIDL-Drive requires high homing efficiency, which has been difficult to achieve in some species (such as mice) so far. For both YLE and fs-RIDL-Drive, female-specific haploinsufficient genes are required, which are rare, or in the case of YLE, X-linked target sites are required.

[0004] For certain use scenarios, if the gene control element can provide relatively complete inhibition of non-localization (self-diffusion), that is, one or several releases can reduce the population in the entire area to a very small number, then the gene control element will be significantly improved. There are currently multiple proposals to achieve this, including the use of a driving Y chromosome or the use of a homing response to knock out genes required for survival or reproduction, or the use of a toxin antidote system including a haploinsufficient gene and a rescue copy thereof. However, these may not be applicable to all species. In addition, the construction of a driving Y chromosome has been proven to be very difficult, and in many species, the incidence of homing may not be high enough, and haploinsufficient genes may be difficult to operate.

[0005] For some use cases, genetic control elements that can provide nonlocalized (self-spreading) partial control would also be improved. This could be useful if the ultimate goal of a control scheme is partial control, or if the ultimate goal is full control but it is desired to be achieved in a stepwise manner as part of a risk mitigation strategy. Currently, no strategies have been proposed that provide self-spreading partial control when the fitness effect and editing rate are ideal (i.e., 0 or 1).

[0006] Therefore, there is a need for a gene control element that does not require high homing efficiency, or the need for female-specific haploinsufficient genes, or sex chromosome expression, and that can provide both localized repression and complete or partial non-localized repression. Summary of the Invention

[0007] As described in the examples, the inventors have developed a novel gene construct that is more efficient at population suppression than SIT, RIDL, or Wolbachia because it can persist in a population for multiple generations. When released without a booster, the gene construct does not require homing and does not require sex-specific genes, and the target site can be autosomal, reducing restrictions on target site selection. The gene construct of the present invention will provide effective suppression of wild populations, and depending on the configuration, it can be confined to the release area or can self-diffuse to other areas, achieving partial or complete suppression. Therefore, potential applications of the gene construct include controlling pest populations that current interventions cannot satisfactorily control.

[0008] Therefore, in a first aspect of the present invention, a genetic construct is provided, which comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence is configured to disrupt a haploid sufficient gene required for survival or reproduction of male and / or female organisms, or is configured to be integrated into or near the disrupted allele of the haploid sufficient gene, and the second nucleotide sequence encodes a genome editor, which produces a dominant lethal mutation or sterility mutation in a target gene expressed in male and / or female organisms, such that: a homozygote of the genetic construct is lethal or sterile, and / or a heterozygote of the dominant lethal mutation or sterility mutation is lethal or sterile.

[0009] In some embodiments, a genetic construct is provided, comprising a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence is configured to disrupt a haploid-sufficient gene required for survival or reproduction of a male and / or female organism, and the second nucleotide sequence encodes a genome editor that produces a dominant lethal mutation or a sterility mutation in a target gene expressed in a male and / or female organism, such that: a homozygote of the genetic construct is lethal or sterile, and / or a heterozygote of the dominant lethal mutation or sterility mutation is lethal or sterile.

[0010] The inventors have unexpectedly discovered that the gene constructs of the present invention can be inserted into the haploid plentiful genes of species members and released into a population to suppress the population in an effective manner over a period of time. Advantageously and preferably, the inventors have found that, depending on the precise configuration of the construct embodiment, the gene constructs can produce three different types of potentially useful effects when released into a target population: localized inhibition, non-localized partial inhibition, or non-localized complete inhibition. These different effects have enormous benefits depending on the species of organism (i.e., pest) whose population is suppressed.

[0011] It is well known to those skilled in the art that if only one working copy of a gene is sufficient to maintain its normal expression and function, then the gene is a haplosufficient (HS) gene. This is because the functional allele of a haplosufficient gene is dominant, while the non-functional allele of a haplosufficient gene is recessive. However, a haploinsufficient (HI) gene is a gene in which a working copy of a gene is not sufficient to maintain its normal expression and function. This is because the non-functional allele of a haploinsufficient gene is actually dominant.

[0012] like Figure 2As shown in a, haploid sufficient gene (HS) is destroyed by the insertion of gene construct (taking Cas9 and gRNA as an example to illustrate).Therefore, gene construct can be configured to destroy the haploid sufficient gene by integrating the gene construct into the haploid sufficient gene at the integration site or the destruction site.For example, in one embodiment, gene construct can introduce premature termination codon into haploid sufficient gene.Or, gene construct can destroy haploid sufficient gene by changing gene (such as the deletion of gene, which can be the complete deletion of gene or the partial deletion of gene).

[0013] The gene construct can be integrated into the center of the haplosufficient gene. The construct can be integrated into the region between the 5' promoter and the 3' end of the haplosufficient gene. The construct can be integrated between exons or introns of the haplosufficient gene.

[0014] However, in some embodiments, the gene construct can be configured to integrate into the disrupted allele of the haploid plentiful gene. Thus, disruption of the haploid plentiful gene can be caused by means other than integration of the gene construct. Preferably, in this embodiment, disruption is caused by the introduction of a knockout mutation, preferably, the knockout mutation is caused by the introduction of a premature stop codon, alternatively by the insertion of a sequence. Alternatively, the knockout mutation can be caused by the partial or complete deletion of the haploid plentiful gene ( Figure 2 d).

[0015] In some embodiments, the genetic construct can be configured to integrate into a position outside of but near the disrupted allele of the haplosufficient gene. Preferably, in this embodiment, the genetic construct is configured to produce an independent modification that disrupts the haplosufficient gene. Thus, in one embodiment, the genetic construct is integrated into a position near the disrupted allele of the haplosufficient gene, preferably within 10, 8, 6, 4, 2, or 1 centimorgan of the disrupted allele of the haplosufficient gene, more preferably within 1 centimorgan of the disrupted allele of the haplosufficient gene ( Figure 2 d).

[0016] A key feature of this novel population suppression method is that the construct is associated with recessive disruption of a haplosufficient gene. One way to achieve this association is to make the construct causally related to the disruption, but this is not the only way. Other methods can accomplish this, including performing two independent operations in close proximity: one to disrupt the haplosufficient gene and the other to integrate the construct. These two operations can be performed in either order. Therefore, references to "a haplosufficient gene disrupted by the construct" and similar expressions encompass both causal and non-causal associations between the disrupted haplosufficient gene and the construct.

[0017] Preferably, the gene into or near which the gene construct is integrated and which is disrupted or which the construct disrupts is haplosufficient, so that an organism heterozygous for one wild-type allele and one disrupted allele in an otherwise wild-type genetic background has normal or near-normal fitness.

[0018] In one embodiment, genetic construct preferably causes the localization suppression of population, promptly is confined to the release area geographically.In order to realize localization suppression, the male and female homozygotes of genetic construct are preferably inviable or sterile.Therefore, in this embodiment, genetic construct is configured to preferably destroy the required haploid sufficient gene of male and female organism survival or breeding, or is configured to be integrated into the allelomorph of the destruction of haploid sufficient gene or near it, makes the male and female homozygotes of this genetic construct be lethal or sterile.Preferably, when genetic construct is integrated into the allelomorph of the destruction of haploid sufficient gene near it, genetic construct is integrated into the allelomorph 10,8,6,4,2 or 1 centimorgan of the destruction of haploid sufficient gene with in, more preferably is integrated into the allelomorph 1 centimorgan of the destruction of haploid sufficient gene with in.

[0019] Furthermore, for localized inhibition, the genome editor preferably produces a dominant lethal or sterile phenotype in female organisms or in both sexes. Thus, in this embodiment, the genome editor preferably produces a dominant lethal or sterile mutation in a target gene expressed in female organisms, or in a target gene expressed in both male and female organisms.

[0020] Thus, in this localized inhibition embodiment, the presence of a genetic construct in or near a haplosufficient gene is associated with recessive lethality or sterility in both sexes (i.e., a recessive lethality or sterility phenotype in both males and females). Preferably, the recessive lethality or sterility is caused by the introduction of a premature stop codon in a haplosufficient gene or by the deletion of all or part of the gene. In a preferred embodiment, the genome editor generates a dominant mutation that is both sex-specific or female-specific, or generates a female-specific dominant mutation that is a male-specific recessive lethal.

[0021] The inventors have generated three molecular configurations of gene constructs capable of achieving localized population suppression, which are shown in Figure 2 a-2c.

[0022] like Figure 2 As shown in Figure 1, in one configuration, the genome editor encoded by the nucleotide sequence of the gene construct targets a site in the wild-type allele of the same haplosufficient gene (which is disrupted by integrating the gene construct). Therefore, in one embodiment, the target gene is the wild-type allele of the disrupted haplosufficient gene.

[0023] Preferably, the genome editor generates a dominant negative or dominant gain-of-function mutation in the wild-type allele of the target gene. Thus, in a preferred embodiment, the genome editor generates a dominant negative or dominant gain-of-function mutation in the wild-type allele of the disrupted haploid sufficient gene.

[0024] Preferably, the genome editor targets a site in the target gene (i.e., the wild-type allele of the haploid plentiful gene) that is located downstream of the integration site. Alternatively, the genome editor can target a site in the haploid plentiful gene that is located upstream of the integration site. In an embodiment where the target site of a genome editor is located upstream of the integration site, the target site on the chromosome containing the gene construct is preferably recoded or removed to protect the chromosome from the influence of dominant mutations.

[0025] Preferably, the genome editor creates a dominant negative or dominant gain-of-function mutation in a female-specific exon of a haplosufficient gene required for male and female organisms, optionally, the haplosufficient gene is a homolog of the Drosophila gene doublesex or fruitless.

[0026] Preferably, the chromosome comprising the construct is not edited because the target site has been modified or removed, or, if it is edited, the dominant negative or dominant gain-of-function mutation is not expressed due to the presence of the gene construct in the haploid sufficient gene, so that the organism comprising a copy of the gene construct and a wild-type allele has a normal or near-normal fitness. However, organisms that are homozygous for the gene construct are preferably non-viable or sterile. In addition, organisms that are heterozygous for the gene construct and the dominant negative or dominant gain-of-function mutation are preferably non-viable or sterile, whether female or sex-neutral. Preferably, when inherited in the absence of a gene construct, a heterozygous dominant lethal mutation or a sterile mutation is lethal or sterile.

[0027] Or, as Figure 2 As shown in b, in another configuration, the genome editor encoded by the nucleotide sequence of the gene construct targets a gene that is tightly linked to the haploid sufficient gene (the gene construct is integrated into or near it). Therefore, in one embodiment, the target gene is a gene that is preferably located near the haploid sufficient gene (i.e., the integration site of the gene construct) of the destruction. Therefore, in a preferred embodiment, the genome editor preferably produces a dominant negative or dominant gain-of-function mutation in the wild-type allele of the target gene located near the haploid sufficient gene of the destruction. Alternatively, in another preferred embodiment, the genome editor produces a knockout mutation in the wild-type allele of the haploid insufficient target gene located near the haploid sufficient gene of the destruction.

[0028] Preferably, when the target gene and the disrupted haploid sufficient gene (i.e., the integration site of the genetic construct) are located near each other, their meiotic recombination rate is less than 5%, less than 4%, less than 3%, less than 2% or less than 1%. Most preferably, the meiotic recombination rate of the target gene and the disrupted haploid sufficient gene is less than 1%.

[0029] Preferably, the target gene and the disrupted haplosufficient gene are inverted. Advantageously, this reduces the frequency of recombination between the two genes.

[0030] Preferably, the copy of the target gene on the same chromosome as the gene construct is modified to make it resistant to the genome editor. For example, if the genome editor produces a dominant negative mutation in a haploinsufficient gene, the resistant chromosome preferably has a deletion of the target site or a deletion of the entire target gene. Alternatively, if the genome editor produces a knockout mutation in a haploinsufficient gene, the resistant chromosome preferably contains a recoded version of the target site so that it is functional but not recognized by the editor. In this case, the genome editor preferably does not stimulate homologous recombination repair.

[0031] Or, as Figure 2 c, in another configuration, the genome editor encoded by the nucleotide sequence of the gene construct can target genes that are distantly linked to the haploid sufficient gene (the gene construct is integrated into or near it). Therefore, in one embodiment, the target gene is a gene that is preferably located at a distance from the haploid sufficient gene (i.e., the integration site of the gene construct) of the destruction. Therefore, in a preferred embodiment, the genome editor produces a dominant negative or dominant gain-of-function mutation in the wild-type allele of the target gene located at a distance from the haploid sufficient gene of the destruction. Alternatively, in another preferred embodiment, the genome editor produces a knockout mutation in the wild-type allele of the haploid insufficient target gene located at a distance from the haploid sufficient gene of the destruction.

[0032] Preferably, when the target gene and the haploid sufficient gene of destruction (i.e., the integration site of the gene construct) are located at a distance from each other, their meiotic recombination rate is greater than 0.5%, greater than 1%, greater than 2%, greater than 3%, greater than 4% or greater than 5%. Most preferably, the meiotic recombination rate of the target gene and the haploid sufficient gene of destruction is greater than 1%. In one embodiment, the target gene and the haploid sufficient gene of destruction are located on different chromosomes. In another embodiment, the target gene and the haploid sufficient gene of destruction are located on the same chromosome.

[0033] like Figure 2As shown in Figure c, the gene construct may also include a rescue copy of the target gene that is not recognized by the genome editor. Therefore, in one embodiment, the gene construct further includes a nucleotide sequence encoding a rescue copy of the target gene. The nucleotide sequence encoding the rescue copy of the target gene is not recognized by the genome editor. For example, in an embodiment where the genome editor is a CRISPR / Cas9-based genome editor, the nucleotide sequence encoding the rescue copy of the target gene is different from the wild-type nucleotide sequence recognized by the gRNA of the genome editor, so the rescue copy is not edited by the Cas9 protein of the genome editor.

[0034] In some embodiments, the nucleotide sequence encoding the target gene rescue copy can be designed to encode an amino acid sequence identical to the wild-type nucleotide sequence or an amino acid sequence equivalent to a functional sequence. In the embodiment in which the genome editor produces a dominant negative mutation, the rescue copy on the construct is preferably configured to dilute the effect of the mutation. Therefore, preferably, the nucleotide sequence encoding the target gene rescue copy is configured to have a higher expression level, to achieve the dilution effect. Alternatively, in the embodiment in which the genome editor produces a haploinsufficient gene knockout, the rescue copy preferably comprises the nucleotide sequence of the protein of the coding knockout. In either case, if there is an allele edited, fitness is rescued, but if there are two alleles edited, fitness is not rescued, so that: for construct, the organism that is heterozygous and carries an allele edited has normal or near-normal fitness, and for construct, the organism that is heterozygous and carries two alleles edited will bear the consequences of the mutation.

[0035] In another embodiment, gene construct preferably causes the complete inhibition of non-localization (self-diffusion), and wherein, the population in whole zone is reduced to very little quantity.In order to realize the complete inhibition of non-localization, the homozygote of gene construct is preferably only for being inviable or sterile in a kind of sex (i.e. male or female).Therefore, in this embodiment, this gene construct is preferably configured to destroy the haploid sufficient gene required for male or female organism survival or breeding, or is configured to be integrated into the allelomorph of the destruction of haploid sufficient gene in or near, makes the male or female homozygote of this gene construct be lethal or sterile.Preferably, when gene construct is integrated into the allelomorph of the destruction of haploid sufficient gene near, gene construct is integrated into the allelomorph 10,8,6,4,2 or 1 centimorgan of the destruction of haploid sufficient gene, more preferably is integrated into the allelomorph 1 centimorgan of the destruction of haploid sufficient gene. Preferably, the genetic construct comprises a nucleotide sequence encoding a genome editor that produces a dominant lethal mutation or a sterility mutation in a target gene expressed in male and / or female organisms.

[0036] like Figure 6 As shown, such non-localized complete inhibition can be achieved in a variety of ways, for example, by inserting a construct into a gene that is required in only one sex, or into an exon that is required in only one sex (due to sex-specific splicing), by incorporating sequences into the construct that ensure that it is spliced ​​out in one sex and not the other, by genome editors that target genes or exons that are required in one or both sexes, and / or by modifying control regions on a gene so that it is expressed in only one sex or that expression is increased to provide partial (sex-specific) protection.

[0037] As described for localized inhibition, the genome editor can target the same haploid abundant gene into which the gene construct is integrated. Thus, in one embodiment, the target gene is the wild-type allele of the disrupted haploid abundant gene. Preferably, the genome editor generates a dominant negative or dominant gain-of-function mutation in the wild-type allele of the target gene. Thus, in a preferred embodiment, the genome editor generates a dominant negative or dominant gain-of-function mutation in the wild-type allele of the disrupted haploid abundant gene.

[0038] In another embodiment, the genome editor can produce a dominant negative or dominant gain-of-function mutation in the wild-type allele of the target gene located near the haploid sufficient gene of the destruction. Alternatively, in another preferred embodiment, the genome editor produces a knockout mutation in the wild-type allele of the haploid insufficient target gene located near the haploid sufficient gene of the destruction. Preferably, when the target gene and the haploid sufficient gene of the destruction (i.e., the integration site of the gene construct) or the haploid insufficient gene are located near each other, their meiotic recombination rate is less than 5%, less than 4%, less than 3%, less than 2% or less than 1%. Most preferably, the meiotic recombination rate of the target gene and the haploid sufficient gene of the destruction is less than 1%.

[0039] Preferably, the chromosome containing the gene construct is not affected by the mutation due to the absence of the target site of the genome editor. Alternatively, the mutation generated by the genome editor is not expressed due to the presence of the gene construct or other mutations at the target site.

[0040] like Figure 6 As shown in a (left side), one method for achieving complete non-localized inhibition is to integrate a gene construct into a female-specific haplosufficient gene and disrupt it. Therefore, in one embodiment, the haplosufficient gene is preferably a female-specific haplosufficient gene. Preferably, the genome editor generates a dominant negative mutation or a dominant gain-of-function mutation in the female-specific haplosufficient gene. Examples of female-specific haplosufficient genes in which dominant sterility mutations have been observed include, but are not limited to, homologs of the Drosophila genes ovo, dorsal, torso, easter, and Toll.

[0041] In another embodiment, the haplosufficient gene can be a male-specific haplosufficient gene. In this embodiment, the genome editor generates a dominant negative mutation or a dominant gain-of-function mutation in the male-specific haplosufficient gene. Examples of male-specific haplosufficient genes in which dominant sterility mutations have been observed include, but are not limited to, homologs of the Drosophila genes betaTub85D and whirligig.

[0042] Alternatively, the haploid plentiful gene of destruction can be expressed in both male and female organisms. Thus, to ensure that the presence of the construct only disrupts the function of the gene in one sex, the gene construct can be integrated into an intron that is spliced ​​in a sex-specific manner, or comprise a sequence that ensures that the construct is sheared out in a sex-specific manner. Thus, although the gene as a whole is essential to both sexes, the presence of the construct only disrupts the function of the gene in one sex.

[0043] For example, Figure 6 a (right side) and Figure 6 Shown in b (left side), the genetic construct is integrated into the female specific exon of the haploid sufficient gene required for both sexes and is destroyed. Therefore, in one embodiment, the haploid sufficient gene of destruction is destroyed at the female specific exon place of this haploid sufficient gene. Two examples of the gene that comprises female specific exon are doublesex (dsx) and fruitless (fru) gene, and they can be from Drosophila species. Therefore, in one embodiment, the haploid sufficient gene of destruction is destroyed at the female specific exon place of doublesex or fruitless gene, or is destroyed at the female specific exon place of the homologue of doublesex or fruitless gene. Or, in another embodiment, the haploid sufficient gene of destruction is destroyed at the male specific exon place of this haploid sufficient gene. The example of the gene that comprises male specific exon includes but is not limited to the homologue of Drosophila gene transformer (tra) and doublesex (dsx). Thus, in one embodiment, the disrupted haplosufficient gene is disrupted at a male-specific exon of a doublesex or transformer gene, or at a male-specific exon of a homolog of a doublesex or transformer gene.

[0044] like Figure 6Shown in b (right side), the haploid plentiful gene of destruction is expressed in male and female organisms, and the genetic construct comprises the sequence that guarantees that this construct is cut out in a sex-specific manner.This can be engineered by using control sequences, and this control sequence limits the sex-specific splicing of natural genes (including but not limited to the homologues of tra, dsx and fru) in the target species.Therefore, in one embodiment, the genetic construct comprises the nucleotide sequence that guarantees that this construct will be cut out on the RNA of male organism.In a preferred embodiment, the genetic construct comprises the nucleotide sequence of the splicing control sequence of the homologue of encoding transformer, doublesex or fruitless gene, and it guarantees that the construct will be cut out on the RNA of male organism.Therefore, gene function is mainly destroyed in female.Or, the genetic construct comprises the nucleotide sequence that guarantees that the construct will be cut out on the RNA of female organism.This can be engineered by using the sequence that guides female-specific splicing, for example, the homologue (Fu etc. .2007Nat.Biotechnol.25:353-7) of Drosophila transformer gene. Therefore, in a preferred embodiment, the gene construct comprises a nucleotide sequence encoding a splicing control sequence of a homolog of a Drosophila transformer gene, which ensures that the construct will be spliced ​​out from the RNA of female organisms. Thus, gene function is primarily disrupted in male organisms. In this embodiment, the mutation generated by the genome editor is preferably a dominant negative mutation in both sexes. Preferably, because the target site has been recoded, the chromosome containing the gene construct is protected from the effects of the genome editor.

[0045] Figure 6 c and 6d show an example of how complete inhibition of self-spreading can be achieved by inserting a construct to disrupt one gene and targeting the genome editor to another gene nearby (<1% recombination), where the target site on the chromosome carrying the construct has been modified and is no longer recognized by the genome editor. Thus, in one embodiment, the genome editor can generate mutations in the wild-type allele of a target gene located near the disrupted haploid sufficient gene, preferably, because the target site has been recoded, the chromosome containing the gene construct is protected from the genome editor. Gene disruption can affect homozygotes of only one sex, and the genome editor can generate dominant lethal mutations or sterility mutations that affect both sexes ( Figure 6c). Thus, preferably, the gene construct is configured to disrupt a haploid-sufficient gene required for survival or reproduction of either male or female organisms, or is configured to integrate into or near a disrupted allele of a haploid-sufficient gene, and the genome editor is configured to produce a dominant lethal mutation or sterility mutation in the target gene expressed in both male and female organisms. Alternatively, the gene disruption may affect homozygotes of both sexes, and the genome editor may also affect both sexes, but for heterozygotes carrying the construct and the mutation, only one sex is affected, which may be achieved by sex-specific enhanced expression of the target gene, which in the heterozygotes negates the effect of the mutation in a sex-specific manner ( Figure 6 d). Therefore, non-localized inhibition can be achieved by modifying the chromosome containing the construct so that the target gene is recoded and therefore not recognized by the genome editor, but still functional, and further comprising a control region so that its expression is enhanced only in one sex, thereby providing partial (sex-specific) protection. Alternatively, the chromosome containing the construct can be modified so that it comprises a second copy of the recoded target gene, the second copy comprising a control region so that it is expressed only in one sex, also providing partial (sex-specific) protection. Therefore, in one embodiment, the gene construct is configured to destroy a haploid sufficient gene required for the survival or reproduction of male and female organisms, or is configured to be integrated into or near the destroyed allele of the haploid sufficient gene, and the genome editor is configured to produce a dominant lethal mutation or sterility mutation in the target gene expressed in male and female organisms. Preferably, in this embodiment, because the target site has been recoded, the chromosome containing the gene construct is protected from the influence of the genome editor, and its expression is enhanced in a sex-specific manner so that only male or female heterozygotes containing the construct and the mutation are affected. Alternatively, the chromosome containing the gene construct contains a second copy of the recoded target gene with control sequences ensuring its expression only in males or females.

[0046] In another configuration, the gene construct can result in a nonlocalized (self-diffusion) partial inhibition ( Figure 8 ). In order to achieve non-localized partial inhibition, the gene construct is preferably recessive sterile or lethal in both sexes or only in one sex. Therefore, in this embodiment, the gene construct is preferably configured to destroy a haploid sufficient gene required for the survival or reproduction of male and / or female organisms, or is configured to be integrated into or near the destroyed allele of the haploid sufficient gene, so that the male and / or female homozygotes of the gene construct are lethal or sterile. In addition, the gene construct comprises a nucleotide sequence encoding a genome editor that produces a dominant lethal mutation or sterility mutation in the target gene expressed in the male and / or female organism.

[0047] If the genetic construct is bisexual (i.e., associated with the disruption of a haplosufficient gene essential for fertility or survival of both male and female organisms), the construct preferably provides dominant protection against the mutation, regardless of its location relative to the construct integration site (i.e., whether the mutation is located in the same gene disrupted by the construct, a linked gene, or an unlinked gene). This is achieved by preferably encoding in the genetic construct a function that negates the effect of the mutation at the RNA level, for example by RNA interference (RNAi) or RNA editing ( Figure 8 , strategies 1 to 4). Therefore, in one embodiment, a gene construct is inserted into and destroys the haploid plentiful gene required for both males and females, or is integrated into or near the allele of the destruction of the haploid plentiful gene required for both males and females, and the genome editor produces a dominant negative mutation of both sexes. In this embodiment, the gene construct includes the nucleotide sequence encoding an RNA editor or an RNA interference module. Preferably, the RNA editor or the RNA interference module recovers the RNA expressed from the mutant gene to a wild-type sequence by RNA editing, or removes the RNA transcribed from the mutant gene by RNA interference (RNAi). This provides dominant protection for mutations.

[0048] If the gene construct is sex-specific (i.e., associated with the destruction of a haploid sufficient gene necessary for male or female fertility or survival), partial inhibition can be achieved in the case of a genome editor that encodes a dominant negative or dominant gain-of-function mutation in the wild-type allele of the same locus. Thus, in one embodiment, the gene construct is preferably inserted into and destroys the haploid sufficient gene required for female organisms, or is integrated into or near the destroyed allele of the haploid sufficient gene required for female organisms, and the genome editor produces a female-specific dominant negative mutation. Alternatively, the gene construct is preferably inserted into and destroys the haploid sufficient gene required for male organisms, or is configured to be integrated into or near the destroyed allele of the haploid sufficient gene required for male organisms, and the genome editor produces a male-specific dominant negative mutation. Alternatively, in another embodiment, the destroyed haploid sufficient gene is destroyed at the female-specific exon of the haploid sufficient gene required for both sexes, and the genome editor produces a female-specific or bisexual dominant negative mutation or gain-of-function mutation. Alternatively, the disrupted haploid sufficient gene is disrupted at the male-specific exon of the haploid sufficient gene required for both sexes, and the genome editor produces a male-specific or bisexual dominant negative mutation or a gain-of-function mutation. Alternatively, in another embodiment, the gene construct is preferably inserted into the haploid sufficient gene required for both males and females, or integrated into or near the disrupted allele of the haploid sufficient gene required for both males and females, but the disrupted haploid sufficient gene is disrupted at the female-specific exon, or the construct comprises a sequence that ensures that it will be sheared off in males, resulting in gene function being primarily disrupted in females. The genome editor preferably produces a bisexual dominant negative mutation. Alternatively, the gene construct is preferably inserted into the haploid sufficient gene required for both males and females, or integrated into or near the disrupted allele of the haploid sufficient gene required for both males and females, but the disrupted haploid sufficient gene is disrupted at the male-specific exon, or the construct comprises a sequence that ensures that it will be sheared off in females, resulting in gene function being primarily disrupted in males. The genome editor preferably generates a female-specific dominant negative mutation, preferably by targeting a female-specific exon of a haploid abundant gene for disruption. Preferably, in some embodiments, the construct provides dominant protection against the mutation regardless of whether the mutation is in cis or in trans (i.e., located in the haploid abundant gene or homologous chromosome into which the construct is integrated). Similarly, this can be achieved if the construct comprises a nucleotide sequence encoding an RNA editor or RNAi module ( Figure 8 , strategies 5 to 13). In a preferred embodiment, the RNA editor or RNAi module is expressed in only one sex ( Figure 8, strategies 15 and 21). In another preferred embodiment, the target site on the chromosome containing the construct is modified or removed so that it is not recognized by the genome editor.

[0049] Alternatively, if the gene construct is inserted into a haploid sufficient gene required for females or required for both males and females, or is configured to be integrated into or near the disrupted allele of the haploid sufficient gene, and the construct comprises a sequence that ensures that it will be sheared off in males, partial inhibition can be achieved, resulting in gene function being primarily disrupted in females. The genome editor preferably produces a dominant negative mutation, a dominant gain-of-function mutation, or a knockout mutation in the wild-type allele of the target gene located near the disrupted haploid sufficient gene. Alternatively, if the gene construct is inserted into a haploid sufficient gene required for males or required for both females and males, or is configured to be integrated into or near the disrupted allele of the haploid sufficient gene, and the construct comprises a sequence that ensures that it will be sheared off in females, partial inhibition can be achieved, resulting in gene function being primarily disrupted in males. The genome editor preferably produces a dominant negative mutation, a dominant gain-of-function mutation, or a knockout mutation in the wild-type allele of the target gene located near the disrupted haploid sufficient gene. Preferably, the construct comprises a nucleotide sequence encoding an RNA editor or an RNAi module.

[0050] Alternatively, partial inhibition can be achieved if the genetic construct is sex-specific (i.e., linked to the disruption of a haplosufficient gene essential for male or female fertility or viability) and encodes a genome editor that produces a dominant negative or dominant gain-of-function mutation in the wild-type allele of the same locus that affects the sex opposite to that in which the gene disruption was made. Figure 8 , strategies 14, 18, 22, and 23). This can be achieved by targeting genes that carry sex-specific exons for both sexes (e.g., doublesex), preferably, the target site on the chromosome carrying the construct is modified so that it is still functional but no longer recognized by the editor. For example, in a preferred embodiment, the disrupted haploid sufficient gene is disrupted at the male-specific exon, resulting in recessive male-specific lethality or sterility, and the genome editor creates a dominant female-specific lethal mutation or sterility mutation in the female-specific exon ( Figure 8 , Strategy 22). Alternatively, the disrupted haploid gene is disrupted at a female-specific exon, resulting in recessive female-specific lethality or sterility, and the genome editor generates a dominant male-specific lethal mutation or sterility mutation at a male-specific exon. Preferably, the target gene for disruption is a homolog of doublesex. Preferably, because the target site has been recoded, the chromosome containing the gene construct is protected from the effects of the genome editor. Figure 10d shows an example of molecular design of strategy 22 using, for example, the doublesex gene.

[0051] Alternatively, partial suppression can be achieved if the genetic construct is sex-specific (i.e., associated with the disruption of a haplosufficient gene essential for male or female fertility or survival) and encodes a genome editor that targets a wild-type allele (meiotic recombination rate <1%) of a target gene located near the disrupted haplosufficient gene, generating a dominant negative mutation, a gain-of-function mutation, or a knockout of a haploinsufficient gene. Preferably, the genetic construct provides dominant protection against the mutation ( Figure 8 , strategies 5 to 13). Preferably, the target site on the chromosome containing the construct has been modified or removed so that it is not recognized by the genome editor. In a preferred embodiment, the RNA editor or RNAi module is expressed in only one sex ( Figure 8 , strategies 15 and 21). In another preferred embodiment, the target gene and the disrupted haploid sufficient gene are inverted to reduce the recombination frequency.

[0052] Alternatively, partial inhibition can be achieved if the genetic construct is sex-specific (i.e., associated with the disruption of a haplosufficient gene essential for male or female fertility or survival), wherein the genetic construct comprises a nucleotide sequence encoding a genomic editor that targets a wild-type allele (meiotic recombination rate <1%) of another gene located near the disrupted haplosufficient gene, resulting in a dominant negative mutation, gain-of-function mutation, or knockout of a haploinsufficient gene that affects the sex opposite to the sex in which the gene disruption was performed. Preferably, the target site on the chromosome containing the construct is modified or removed so that it is no longer recognized by the editor ( Figure 8 , strategies 14, 18, 22 and 23). Preferably, the gene construct comprises sequences that ensure that it will be sheared out in a sex-specific manner. Preferably, the target gene and the disrupted haploid plentiful gene are inverted to reduce the recombination frequency.

[0053] Alternatively, if the genetic construct is sex-specific (i.e., associated with the disruption of a haplosufficient gene essential for male or female fertility or viability) and encodes a genome editor that targets a wild-type allele of another gene located distal to the disrupted gene (meiotic recombination rate >1%), a dominant negative mutation, gain-of-function mutation, or knockout of a haploinsufficient gene that affects the sex opposite to that affected by the disrupted gene is generated ( Figure 8 Strategies 14 and 22, and Figure 9 Alternatively, genome editors can generate mutations in the sex opposite to the sex affected by the disruption ( Figure 8, strategies 28 and 30). Preferably, the genetic construct comprises a nucleotide sequence encoding a recoded version of the edited gene, rendering it resistant to the genome editor while also restoring function in individuals carrying a single copy of the dominant edit. Preferably, the genetic construct comprises a sequence that ensures it will be excised in a sex-specific manner.

[0054] The inventors have generated other molecular configurations of gene constructs that can achieve localized population inhibition, non-localized (self-diffusion) complete inhibition, and non-localized (self-diffusion) partial inhibition, such as Figure 11 shown.

[0055] like Figure 11 As shown, the construct is configured to integrate into a location outside the haplosufficient gene (near it or distantly linked to it) and is designed to induce a mutation in the haplosufficient gene that results in recessive sterility or lethality, for example, by including a second gRNA in the construct that targets the haplosufficient gene.

[0056] Therefore, in a second aspect of the present invention, a genetic construct is provided, which comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence is configured to be integrated into a position outside a haploin sufficient gene required for survival or reproduction of male and / or female organisms, and the second nucleotide sequence encodes: (i) a first genome editor, which produces a dominant lethal mutation or a sterility mutation in a target gene expressed in male and / or female organisms, so that heterozygotes of the dominant lethal mutation or sterility mutation are lethal or sterile; and (ii) a second genome editor, which produces a recessive lethal mutation or a sterility mutation in the haploin sufficient gene, so that homozygotes of the recessive lethal mutation or sterility mutation are lethal or sterile.

[0057] Preferably, heterozygotes for a dominant lethal or sterility mutation are lethal or sterile when inherited in the absence of the genetic construct.

[0058] A position outside the haplosufficient gene can be near the haplosufficient gene or remotely linked to the haplosufficient gene. A position near the haplosufficient gene can be within 10, 8, 6, 4, 2, or 1 centimorgan of the haplosufficient gene, more preferably within 1 centimorgan of the haplosufficient gene. Alternatively, a position outside the haplosufficient gene can be located on a different chromosome than the haplosufficient gene (i.e., unlinked).

[0059] Preferably, the haploid plentiful gene is located downstream of the integration site of the gene construct. Alternatively, the haploid plentiful gene can be located upstream of the integration site of the gene construct. Alternatively, the haploid plentiful gene can be located on a chromosome different from the integration site of the gene construct.

[0060] like Figure 11 As shown in a, the genetic construct can encode i) a first genome editor that targets the wild-type allele of a haplosufficient gene, resulting in dominant lethality or sterility in both sexes, and ii) a second genome editor that targets an allele linked to the construct and located upstream of the target site of the first genome editor, generating a premature stop codon that results in recessive sterility or lethality in both sexes.

[0061] Therefore, preferably, the first genome editor generates a dominant negative or dominant gain-of-function mutation in the wild-type allele of the target gene, which results in dominant lethality or sterility in males and females.

[0062] For localized inhibition, the first genome editor preferably produces a dominant lethal or sterility phenotype in female organisms or in both sexes of the organism. Thus, in a preferred embodiment, the first genome editor produces a dominant lethal mutation or sterility mutation in a target gene expressed in female organisms, or in a target gene expressed in both male and female organisms.

[0063] Preferably, the second genome editor generates a premature stop codon in an allele of the target gene that is linked to the gene construct and located upstream of the target site of the first genome editor. Thus, in a preferred embodiment, the second genome editor generates a premature stop codon in a haplosufficient gene. Preferably, the second genome editor generates a premature stop codon in an allele of the haplosufficient gene that is linked to the gene construct and located upstream of the target site of the first genome editor.

[0064] This premature stop codon causes recessive sterility or lethality in males and females. The presence of the premature stop codon prevents expression of the downstream dominant negative mutation, so individuals that inherit one copy of the construct and its associated stop codon and carry a wild-type allele have near-normal fitness (although they would be nonviable or sterile if the construct and the associated stop codon were homozygous).

[0065] By recoding the allele linked to the construct so that it remains functional but can be targeted by the genome editor, while the wild-type allele is unaffected, specificity of the allele linked to the construct can be achieved. Thus, in one embodiment, the allele of the target gene linked to the gene construct has been recoded so that it can be targeted by the second genome editor. Preferably, the wild-type allele is not recognized by the second genome editor.

[0066] like Figure 11As shown in Figures 11b and 11c, the genetic construct can be configured to induce a recessive sterility or lethal mutation in a second gene that is independent of the gene targeted to induce a dominant sterility or lethal mutation.

[0067] Thus, in one embodiment, the second genome editor creates a recessive lethal or sterility mutation in a haplosufficient gene. Preferably, the first genome editor creates a dominant lethal or sterility mutation in a haploinsufficient gene or any gene that may create a dominant lethal or sterility mutation in both males and females or only in females. The haplosufficient and / or haploinsufficient genes may be located at any distance from the genetic construct. In one embodiment, the gene targeted by the first genome editor is a haploinsufficient gene. Alternatively, the gene targeted by the first genome editor may be any gene that may create a dominant lethal or sterility mutation in both males and females or only in females.

[0068] In this embodiment, the construct may be linked to a haploinsufficient gene or any gene that may produce a dominant lethal mutation or sterility mutation in males and females or in females only ( Figure 11 b) or non-chain ( Figure 11 c). Thus, in one embodiment, the construct is linked to the gene targeted by the first genome editor (i.e., a haploinsufficient gene, or any gene that may produce a dominant lethal mutation or infertility mutation in both males and females or only in females). Those skilled in the art will understand that "linked" means that the construct and the haploinsufficient gene or any gene that may produce a dominant lethal mutation or infertility mutation in both males and females or only in females are located on the same chromosome. In one embodiment, the construct and the haploinsufficient gene or any gene that may produce a dominant lethal mutation or infertility mutation in both males and females or only in females are located within 10, 8, 6, 4, 2 or 1 centimorgan of each other.

[0069] Alternatively, in another embodiment, the gene construct is unlinked to the gene targeted by the second genome editor (i.e., a haploinsufficient gene, or any gene that may produce a dominant lethal mutation or infertility mutation in males and females or only in females). Those skilled in the art will understand that unlinked means that the construct is located on a different chromosome from the haploinsufficient gene or any gene that may produce a dominant lethal mutation or infertility mutation in males and females or only in females.

[0070] like Figure 11As shown in Figure c, the gene construct may further comprise a rescue copy of the target gene that is not recognized by the genome editor. Therefore, in one embodiment, the gene construct further comprises a nucleotide sequence encoding a rescue copy of the target gene. The nucleotide sequence encoding the rescue copy of the target gene is not recognized by the genome editor. For example, in an embodiment where the genome editor is a CRISPR / Cas9-based genome editor, the nucleotide sequence encoding the rescue copy of the target gene is different from the wild-type nucleotide sequence recognized by the gRNA of the genome editor, so that the rescue copy is not edited by the Cas9 protein of the genome editor.

[0071] However, the nucleotide sequence encoding the target gene rescue copy can also be designed to encode an amino acid sequence identical to the wild-type nucleotide sequence or an amino acid sequence that is functionally equivalent. In an embodiment in which the genome editor produces a dominant negative mutation, the rescue copy on the construct is preferably configured to dilute the mutation effect. Therefore, preferably, the nucleotide sequence encoding the target gene rescue copy is configured to have a higher expression level, to achieve the dilution effect. Alternatively, in an embodiment in which the genome editor produces a knockout of a haploinsufficient gene, the rescue copy preferably comprises a nucleotide sequence encoding the protein of the knockout. In either case, if there is an edited allele, fitness is rescued, but if there are two edited alleles, fitness is not rescued, so that: an organism that is heterozygous for a construct and carries an edited allele has a normal or near-normal fitness, while an organism that is heterozygous for a construct and carries two edited alleles will suffer the consequences of the mutation.

[0072] Advantageously, based on this aspect of the invention, all fitness costs incurred by the constructs could potentially be suppressed by using Cas9 inhibitors. This would allow transgenic organisms to be maintained as pure-breeding lines, thereby reducing breeding costs and time.

[0073] Depending on the insertion site of the editor (or the location of the associated disruption), all three different types of repression can be achieved by targeting the female-specific exons of the doublesex homolog to generate dominant negative mutations. Insertion (or disruption) of the 5' region required for both sexes results in localized repression ( Figure 2 a right); insertion (or disruption) of the female-specific exon results in nonlocalized complete inhibition ( Figure 6 a right); insertion (or disruption) of the male-specific exon results in a nonlocalized partial inhibition ( Figure 10 d).

[0074] Organisms suitable for targeting using the gene constructs of the present invention include disease vectors, agricultural pests or harmful invasive species. Preferably, the types of pest species include but are not limited to arthropods, other invertebrates (such as mussels), mammals, other vertebrates (such as fish) and weeds.

[0075] Arthropods suitable for targeting using the gene drive gene constructs of the present invention include insects, arachnids, myriapods, or crustaceans. Preferably, the arthropod is an insect. Preferably, the arthropod (most preferably an insect) is a disease vector or pest (e.g., an agricultural pest) that can infect, harm, or kill animals or plants of agricultural value, for example, Anopheles species, Aedes species (as disease vectors), Mediterranean fruit fly (Ceratitis capitata), or Drosophila species (as agricultural pests).

[0076] Alternatively, the insect may be a mosquito. Preferably, the mosquito is a mosquito of the subfamily Anophelesinae. Preferably, the mosquito is selected from the group consisting of Anopheles gambiae, Anopheles coluzzi, Anopheles merus, Anopheles melas, Anopheles arabiensis, Anopheles quadriannulatus, Anopheles stephensi, Anopheles arabiensis, Anopheles funestus, Anopheles albimanus, Anopheles darlingi, and Anopheles sinensis. Alternatively, the mosquito may be a mosquito of the genus Aedes, preferably Aedes aegypti or Aedes albopictus. Alternatively, the mosquito may be a mosquito of the genus Culex, preferably Culex pipiens or Culex quinquefasciatus.

[0077] Alternatively, the insect may be a fruit fly of the family Tephritidae, preferably the Mediterranean fruit fly (Mediterranean fruit fly), a Bactrocera spp. (including B. oleae, B. dorsalis, tryoni) or an Anastrepha spp. (including A. grandis, A. ludens, A. obliqua, A. suspensa).

[0078] Alternatively, the insect may be a sand fly, preferably a species that transmits disease. Preferably, the sand fly may be a Phlebotomus spp. or a Lutzomyia spp., preferably L. longipalpis.

[0079] Alternatively, the insect may be another Diptera insect, preferably Glossina spp. (tsetse flies), Rhagoletis pomonella (apple maggots), Drosophila suzukii (spotted-wing drosophila), Cochliomyia hominivorax (screwworm maggots) or Lucilia cuprina (Australian sheep blowfly).

[0080] Alternatively, the insect may be an insect of the order Lepidoptera, preferably an insect of the order Lepidoptera that is an agricultural pest. Preferably, the lepidopteran insect is selected from one of the following: Pectinophora gossypiella (pink bollworm), Lymantriadispar (sponge moth), Epiphyas postvittana (light brown apple moth), Lobesia botrana (European grape moth), Cydia pomonella (apple leafroller), Synanthedon myopaeformis (apple clearwing moth), Plutella xylostella (diamondback moth), Spodoptera frugiperda (fall armyworm), Phthorimaea absoluta (formerly known as Tuta absoluta; tomato pinworm), Chilopartellus (stem borer), Helicoverpa armigera (Old World bollworm) and Helicoverpa zea)(corn earworm).

[0081] Alternatively, the insect may be a Coleopteran insect, preferably an agricultural pest Coleopteran insect. Preferably, the Coleopteran insect is selected from one of the following: Rhynchophorus ferrugineus (red palm weevil), Hypothenemus hampei (coffee berry borer), Sternochetus frigidus (mango pulp weevil), Sitona obsoletus (clover root weevil).

[0082] Alternatively, the insect may be a mealybug, preferably a mealybug that is an agricultural pest. Preferably, the mealybug is selected from the group consisting of Phenacoccus manihoti (cassava mealybug) and Phenacoccus solenopsis (mealybug).

[0083] Alternatively, the organism may be another arthropod, preferably a harmful invasive arthropod, such as Pacifastacus leniusculus (signal crayfish).

[0084] Alternatively, the organism may be a gastropod, preferably a gastropod that is an intermediate host of a parasite of humans or other vertebrates, such as a member of the genus Bulinus spp., Biomphalaria spp., or Oncomelania spp. Alternatively, the gastropod may be a harmful invasive species, preferably Crepidula fornicata (common slipper snail) or Pomacea canaliculata (golden apple snail).

[0085] Alternatively, the organism may be a bivalve, preferably a harmful invasive bivalve, such as Dreissena polymorpha (zebra mussel), Limnoperna fortunei (golden mussel), or Corbicula fluminea (invasive Asian clam).

[0086] Alternatively, the organism may be a fish, preferably an invasive fish. Preferably, the fish is Cyprinus carpio (common carp), Petromyzon marinus (marine lamprey), Salvelinus fontinalis (brook trout) or Pseudorasbora parva (stone carp).

[0087] Alternatively, the organism may be an amphibian, preferably a harmful invasive amphibian, such as Rhinella marina (cane toad) or Lithobates catesbeianus (American bullfrog).

[0088] Alternatively, the organism may be a mammal, preferably a harmful invasive mammal. Preferably, the mammal is Mus musculus (house mouse), Mus domesticus (house mouse), Rattus norvegicus (brown rat), Rattus rattus (black rat), Rattus exulans (Polynesian rat), Oryctolagus cuniculus (common rabbit), Felis silvestris catus (wild cat), Sciurus carolinensis (grey squirrel), or Trichosurus vulpecula (common brushtail possum).

[0089] Alternatively, the organism may be a plant, preferably a weed or harmful invasive plant. Preferably, the plant is selected from the group consisting of: Amaranthus palmeri, Amaranthus tuberculatus, Alopecurus myosuroides, Lolium rigidum, Kochia scoparia, Centaurea maculosa, Lantana camara, Ambrosia artemisiifolia, and Eragrostis plana.

[0090] In some embodiments, organism comprises the allele of the destruction of haploid plentiful gene.Therefore, in some embodiments, the destruction of haploid plentiful gene is caused by the mode other than integrating gene construct.Preferably, destruction is caused by the introduction of knockout mutation, preferably, knockout mutation is caused by the introduction of premature termination codon, alternatively caused by the insertion of sequence.Therefore, in some embodiments, organism comprises the knockout mutation of haploid plentiful gene, preferably, organism comprises premature termination codon.Or, knockout mutation can be caused by the partial or complete deletion of haploid plentiful gene.Therefore, in some embodiments, organism comprises the partial or complete deletion of haploid plentiful gene.

[0091] As described above, genome editors can target haplosufficient genes into or near which the gene construct is integrated. Specifically, for localized inhibition, the disrupted gene needs to be haplosufficient and essential for the survival and reproduction of both sexes. Therefore, in one embodiment, the target gene is the wild-type allele of the disrupted haplosufficient gene.

[0092] In another embodiment, the target gene can be any other gene that can produce dominant negative or dominant gain-of-function mutation. Dominant negative mutations include mutations that cause interference with the normal function of wild-type allele proteins. Dominant gain-of-function mutations are mutations that cause proteins to obtain new functions. Therefore, in one embodiment, the target gene can be a haploid plentiful gene. The target haploid plentiful gene can be a gene different from the haploid plentiful gene (which is destroyed by the integration of the gene construct).

[0093] One common way to generate dominant negative mutations is through "multimer toxicity." For example, if a protein normally functions as a dimer, and wild-type and mutant protein molecules are equally common and randomly associated, then only 1 / 4 of the dimers will be composed of two wild-type molecules and function correctly, which may be too few for correct function. Therefore, in one embodiment, the target gene can encode a protein that functions as a multimer.

[0094] Classic examples of gene classes that can be mutated to produce dominant negative mutations include homodimeric membrane receptors and transcription factors.Thus, preferably, the target gene encodes a transcription factor or a membrane-bound protein, preferably a homodimeric membrane receptor.

[0095] In embodiments where the organism is an insect, the target gene can be doublesex (dsx), or a homolog of the doublesex gene. Knockout mutations of doublesex are bisexually sterile, while stop codons introduced into female-specific exons can be dominant female sterile. More specifically, the 5' region of this gene encodes a DNA binding domain expressed in both sexes, and knockout in this region results in homozygous intersex and sterility in both sexes, while heterozygotes have more or less normal fertility. Therefore, in one embodiment, a genome editor is inserted into and disrupts the 5' region of the doublesex gene, where this region encodes a DNA binding domain expressed in both male and female organisms.

[0096] There are female-specific exons downstream of this region, in which premature stop codons can lead to dominant female infertility. Therefore, in a preferred embodiment, the genome editor targets the female-specific exons of the doublesex gene. Preferably, the genome editor introduces premature stop codons into the female-specific exons of the doublesex gene. For example, this can be achieved by base editing, reverse leader editing, introduction of insertion / deletion (indel) mutations, or stimulation of homing of stop codons introduced into the haplotype of the present invention by researchers. This will cause the wild-type allele to mutate into a dominant female-specific infertile allele. Due to the upstream stop codon caused by the insertion of the construct, the dominant female sterility edit on the chromosome containing the construct will not be expressed.

[0097] Alternatively, any gene that is haplosufficient but editable to produce a dominant sterile or lethal phenotype can be used. In one embodiment, the haplosufficient gene can be selected from the group consisting of homologs of the following Drosophila genes: 5-HT2A, Antp, BicD, cact, chic, Col4a1, crn, cype, dare, dl, dpp, Fs(2)Ket, gro, hb, hh, hop, Hsc70-3, Hsc70-4, ken, l(1)10Ad, l(1)10Ae, l(2)25 Ca, l(2)40Ff, l(2)46Fb, l(2)DTS18SP, l(2)DTS19, l(2)DTS20, l(2)DTS6, l(2)DTS8, l(2)D TS9, l(2)M167, lt, M(3)80, nos, Prosbeta6, puc, rl, Scr, snf, stmA, Sxl, tkv, Tl, tor and wupA.

[0098] Among the above genes, 15 genes have invalid or loss-of-function recessive lethal alleles. Therefore, in a preferred embodiment, the haploid sufficient gene can be selected from: Antp, BicD, cact, chic, dpp, gro, hh, hop, puc, rl, Scr, snf, Sxl, tkv and wupA.

[0099] In another embodiment, the target gene is a haploinsufficient gene. Preferably, in this embodiment, the mutation generated by the genome editor results in the knockout of the haploinsufficient gene. This is because for a haploinsufficient gene, only one active allele is insufficient for normal function. In one embodiment, the haploinsufficient gene is a haplolethal gene. A haplolethal gene is a special type of haploinsufficient gene in which individuals with only one active gene copy die early in development (before reproduction).

[0100] For example, Drosophila melanogaster contains at least 43 haploid lethal or haploid sterility genes. Most of these encode protein components of the cytoplasmic ribosome (Rp genes) or translation initiation factors (eIF genes) and are associated with Minute syndrome (a phenotypic feature set including short and thin bristles and developmental delay). Therefore, in a preferred embodiment, the genome editor targets homologs of cytoplasmic ribosomal proteins or translation initiation factors.

[0101] In another embodiment, the target gene can be a haploinsufficient gene, including genes encoding the muscle components actin (Act88F), myosin (Mhc and Mlc2), and tropomyosin (Tm2), and a group of tightly linked muscle-related genes regulated by a haplolethal sequence in the intron of the troponin I (wupA) gene. This gene subset can also include Hdl, which can correspond to troponin T (up). Other haploinsufficient gene subsets encode homeodomain proteins (Abd-B, Dll, Scr, and Ubx), Notch pathway components (Dl, H, and N), Polycomb family repressors (Pc and Pcl), apoptosis regulators (lok and p53), and melanin biosynthetic enzymes (b and e).

[0102] In some embodiments, the disrupted gene, while essential in nature, is not essential in the laboratory for the purpose of cultivating homozygous, pure-breeding lines. Thus, the gene disruption can be auxotrophic and can be compensated for by dietary supplementation. Thus, in one embodiment, the disrupted gene is involved in the biosynthesis of purines, pyrimidines, or fatty acids, or can be rescued by dietary supplementation with these factors, fructose, or linolenic acid. The target gene can be a homolog of the Drosophila gene rudimentary, rudimentary-like, Dhod, ade2, ade3, ade4, ade5, bur, Pgd, or SREBP.

[0103] The gene construct comprises a nucleotide sequence encoding a sequence-specific genome editor that edits naturally occurring sequences in the genome to produce lethal mutations or sterile mutations. Several genome editors are well known to those skilled in the art. The genome editor can be selected from: a transcription activator-like effector nuclease (TALEN) genome editor, a zinc finger nuclease (ZFN) genome editor, and a CRISPR-based genome editor. Preferably, the genome editor is a CRISPR-based genome editor, most preferably a CRISPR-Cpf1 or CRISPR-Cas9-based genome editor. However, it should be understood that other nucleases used in CRISPR-based genome engineering methods are known and can be used according to the present invention.

[0104] Therefore, in one embodiment in which the genome editor is a CRISPR-based genome editor, the genome editor comprises a first nucleotide sequence capable of hybridizing with the target gene. Preferably, the first nucleotide sequence capable of hybridizing with the target gene is a guide RNA (gRNA). In a preferred embodiment, the genome editor comprises a nucleotide sequence encoding at least one gRNA, preferably two gRNAs or three gRNAs. In a preferred embodiment, the genome editor comprises a nucleotide sequence encoding two gRNAs. Advantageously, more than one gRNA can improve editing efficiency and / or prevent the evolution of resistance.

[0105] Preferably, the CRISPR-based genome editor further comprises a second nucleotide sequence encoding a CRISPR nuclease (preferably Cpf1 or Cas9 nuclease, most preferably Cas9 nuclease), or a derivative thereof, to allow DNA nicking, base editing, lead editing or other types of editing. CRISPR nucleases and sequences encoding nucleotides are known in the art. The first nucleotide sequence and the second nucleotide sequence can be located on independent nucleic acid molecules to form two gene constructs, which act in tandem (i.e., trans form) as the gene construct of the present invention. However, preferably, the first nucleotide sequence and the second nucleotide sequence are located on the same nucleic acid molecule or form part of the same nucleic acid molecule, thereby producing the gene construct of the present invention. Preferably, the second nucleotide sequence encoding the nuclease is located 5' of the first nucleotide sequence encoding a nucleotide sequence capable of hybridizing with the target gene.

[0106] The portion of the nucleotide sequence that is capable of hybridizing to the target gene (i.e., the guide RNA) is called a protospacer. In order for the nuclease to work, it also requires a specific protospacer adjacent motif (PAM), which varies depending on the bacterial species of the nuclease-encoding gene. The most commonly used Cas9 nuclease recognizes a PAM sequence of NGG, which is located immediately downstream of the target sequence on the non-target strand of genomic DNA. The recognition of the PAM by the nuclease is thought to destabilize the adjacent sequence, allowing the guide RNA to probe the sequence and lead to RNA-DNA pairing when a matching sequence is present. The PAM is not present in the guide RNA sequence, but is required immediately downstream of the target site in the genomic DNA.

[0107] Those skilled in the art will appreciate that the nucleotide sequence capable of hybridizing to the target gene (i.e., the guide RNA) may also comprise a CRISPR nuclease binding sequence (preferably a Cpf1 or Cas9 nuclease binding sequence, most preferably a Cas9 nuclease binding sequence). The CRISPR nuclease binding sequence forms a secondary binding structure, such as a hairpin loop, that complexes with the nuclease. The PAM on the host genome is recognized by the nuclease.

[0108] The CRISPR-based genome editor further comprises at least one promoter sequence that drives the expression of the first nucleotide sequence and the second nucleotide sequence. In other words, the expression of the first nucleotide sequence and the second nucleotide sequence is controlled by the same promoter. Alternatively, the CRISPR-based genome editor may comprise at least two promoter sequences so that the expression of the first nucleotide sequence and the second nucleotide sequence is controlled by independent promoters. Therefore, in one embodiment, the editor comprises a first promoter sequence operably linked to the first nucleotide sequence, and a second promoter sequence operably linked to the second nucleotide sequence. The first promoter sequence and the second promoter sequence can be any promoter sequence suitable for expression in an organism and are known to those skilled in the art. Therefore, the guide RNA is preferably expressed under the control of the first promoter, and the nuclease is expressed under the control of the second promoter.

[0109] Preferably, the first promoter is a polymerase III promoter, most preferably a polymerase III promoter without a 5' end cap or a 3' end poly A tail. More preferably, the promoter is a U6 promoter.

[0110] Preferably, the genome editor comprises a control sequence that ensures that the editor is active in the male and / or female reproductive system, so that most offspring inherit lethal mutations or infertility mutations. Therefore, in one embodiment, the genetic construct further comprises a nucleotide sequence comprising a control sequence that ensures that the genome editor is active in the male and / or female reproductive system. More preferably, the genetic construct comprises a first nucleotide sequence and a second nucleotide sequence, and the first nucleotide sequence and the second nucleotide sequence comprise a first control sequence and a second control sequence. Preferably, the first nucleotide sequence and the second nucleotide sequence comprising the first control sequence and the second control sequence are located on the flanks of the genome editor. Preferably, the control sequence includes but is not limited to a promoter, an enhancer, a terminator or other regulatory sequence. Alternatively, the genome editor may be active in tissues other than the reproductive system.

[0111] Control sequences for specifying gene expression in the germline can be selected from native genes that exhibit germline expression in the target organism. Preferably, control sequences include homologs of the Drosophila genes vasa, nanos, zpg, exu, mei-W68 (spo11), and betaTubulin. Alternatively, these genes can be found by performing RNA sequencing (RNAseq) experiments on the target organism, comparing gene expression in germline and somatic tissues. For mouse, rat, and other mammalian target species, useful sequences can be found in Lai et al. (2022: https: / / www.biorxiv.org / content / 10.1101 / 2022.08.30.505951v1).

[0112] In one embodiment, the gene construct comprises a nucleotide sequence encoding a fluorescent marker. For example, the nucleotide sequence can encode green fluorescent protein (GFP). Advantageously, the fluorescent marker can be used to track the presence of the construct.

[0113] Preferably, the first nucleotide sequence encoding a nucleotide sequence that hybridizes to the target gene (i.e., the guide RNA) targets the editor to the target gene when transcription occurs.

[0114] Preferably, the genetic construct is inserted into the genome by homologous recombination or homology-directed repair. Thus, preferably, the nucleotide sequence configured to disrupt the haploid plentiful gene comprises a nucleotide sequence capable of hybridizing with the haploid plentiful gene. Preferably, the nucleotide sequence configured to disrupt the haploid plentiful gene is substantially complementary to or homologous to at least one region of the haploid plentiful gene, such that homologous recombination occurs therebetween. Preferably, the genetic construct comprises nucleotide sequences flanking the nucleotide sequence encoding the genome editor, wherein each flanking sequence is substantially complementary to or homologous to at least one region of the haploid plentiful gene, such that homologous recombination occurs therebetween.

[0115] Preferably, the or each nucleotide sequence configured to disrupt the haplosufficient gene is substantially complementary to or homologous to a nucleotide sequence of the haplosufficient gene such that the genetic construct integrates into the genome.

[0116] In another embodiment, the gene construct is inserted into the genome by recombinase-mediated cassette exchange (a technique known to those skilled in the art). Therefore, preferably, the gene construct further comprises an integrase attachment site (preferably an attB integrase attachment site) that is located on either side of the nucleotide sequence encoding the genome editor.

[0117] In a preferred embodiment, the gene construct is introduced into a genome comprising a docking construct, wherein the docking construct comprises an integrase attachment site (preferably an attP integrase attachment site) flanked by 5' and 3' homology arms homologous to genomic sequences flanking the insertion / integration site (whether located within or near a haplosufficient gene), such that the docking construct is introduced into the genome by homology directed repair. The gene construct is preferably inserted into the genome by recombinase-mediated cassette exchange, wherein the docking construct is inserted into the genome by an integrase (preferably an attP integrase attachment site) flanked by 5' and 3' homology arms homologous to genomic sequences flanking the insertion / integration site (whether located within or near a haplosufficient gene). Integrase), the docking construct is exchanged for the gene construct.

[0118] For example, the gene construct can be a plasmid, a cosmid or a phage and / or a viral vector. These recombinant vectors are very useful in the delivery system for transforming cells of the present invention. The nucleotide sequence can be preferably a DNA sequence. The gene construct can also include a variety of other functional elements, including a suitable regulatory sequence for controlling its expression after the gene construct is introduced into the host cell. The construct can also include a regulator or enhancer to control the expression of the required construct elements. Tissue-specific enhancer elements (such as promoter sequences) can be used for further regulating and controlling the expression of the construct in organism cells.

[0119] In a third aspect, there is provided a use of the genetic construct of the first aspect for disrupting a haplosufficient gene required for the survival or reproduction of male and / or female organisms, or for integrating into or near a disrupted allele of a haplosufficient gene, rendering the homozygote of the genetic construct lethal or sterile, and generating a dominant lethal mutation or sterile mutation in a target gene expressed in male and / or female organisms, rendering heterozygotes of the dominant lethal mutation or sterile mutation lethal or sterile, and / or rendering male and / or female organisms comprising the dominant lethal mutation or sterile mutation infertile.

[0120] In some embodiments, the disrupted allele of a haplosufficient gene comprises a knockout mutation, optionally, the knockout mutation results from the introduction of a premature stop codon, or alternatively, the knockout mutation can result from the partial or complete deletion of a haplosufficient gene.

[0121] In some embodiments, there is provided a use of the gene construct of the first aspect for disrupting a haplosufficient gene required for survival or reproduction of a male and / or female organism, rendering the homozygote of the gene construct lethal or sterile, and generating a dominant lethal mutation or sterile mutation in a target gene expressed in the male and / or female organism, rendering the heterozygote of the dominant lethal mutation or sterile mutation lethal or sterile, and / or rendering the male and / or female organism comprising the dominant lethal mutation or sterile mutation infertile.

[0122] In a fourth aspect, there is provided a use of the gene construct of the second aspect for integration into a position outside a haplosufficient gene required for survival or reproduction of male and / or female organisms, and for generating a dominant lethal mutation or sterility mutation in the target gene expressed in the male and / or female organisms, rendering heterozygotes of the dominant lethal mutation or sterility mutation lethal or sterile, and for generating a recessive lethal mutation or sterility mutation in a haplosufficient gene expressed in the male and / or female organisms, rendering homozygotes of the recessive lethal mutation or sterility mutation lethal or sterile.

[0123] In a fifth aspect, there is provided a method of producing a genetically modified organism, the method comprising introducing into the organism the genetic construct of the first aspect or the second aspect.

[0124] In a sixth aspect, a method for producing a genetically modified organism is provided, the method comprising introducing a genetic construct into the organism, the genetic construct comprising a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence being configured to disrupt a haploid sufficient gene required for survival or reproduction of the male and / or female organism, or being configured to integrate into or near the disrupted allele of the haploid sufficient gene, the second nucleotide sequence encoding a genome editor, which genome editor produces a dominant lethal mutation or a sterility mutation in the target gene of the male and / or female organism, such that: a homozygote of the genetic construct is lethal or sterile, and / or a heterozygote of the dominant lethal mutation or sterility mutation is lethal or sterile.

[0125] In some embodiments, the disrupted allele of a haplosufficient gene comprises a knockout mutation, optionally, the knockout mutation results from the introduction of a premature stop codon, or alternatively, the knockout mutation can result from the partial or complete deletion of a haplosufficient gene.

[0126] In some embodiments, a method for producing a genetically modified organism is provided, the method comprising introducing a genetic construct into the organism, the genetic construct comprising a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence being configured to disrupt a haploid-sufficient gene required for survival or reproduction of the male and / or female organism, the second nucleotide sequence encoding a genome editor, the genome editor generating a dominant lethal mutation or a sterility mutation in a target gene of the male and / or female organism, such that: a homozygote of the genetic construct is lethal or sterile, and / or a heterozygote of the dominant lethal mutation or sterility mutation is lethal or sterile.

[0127] In a seventh aspect, a method for producing a genetically modified organism is provided, the method comprising introducing a genetic construct into the organism, the genetic construct comprising a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence being configured to integrate into a position outside a haploin sufficient gene required for survival or reproduction of the male and / or female organism, the second nucleotide sequence encoding: (i) a first genome editor, the first genome editor generating a dominant lethal mutation or a sterility mutation in a target gene expressed in the male and / or female organism; and (ii) a second genome editor, the second genome editor generating a recessive lethal mutation or a sterility mutation in the haploin sufficient gene, rendering a homozygote for the recessive lethal mutation or sterility mutation lethal or sterile.

[0128] The genome editing method or technique can be performed in vivo, in vitro or ex vivo.Preferably, the haplosufficient gene, target gene, genetic construct and organism of the third to seventh aspects are as defined in the first or second aspect.

[0129] The gene construct is introduced directly into the host cell of the organism (preferably the host cell of the organism in the embryo of the organism) by a suitable means (e.g., direct endocytic uptake). The construct can be introduced directly into the cells of the host organism (e.g., mosquito) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion, or ballistic bombardment. Alternatively, a particle gun can be used to introduce the construct of the present invention directly into the host cell.

[0130] The inventor has also unexpectedly found that the second construct can be used to increase the frequency of the first gene construct in the population. Therefore, the method of the fifth, sixth or seventh aspect can also include introducing the second gene construct into the organism, the second gene construct comprising a nucleotide sequence that is configured to increase the frequency of the first gene construct. In this embodiment, the first gene construct is a gene construct that is associated with the destruction of a haploid plentiful gene or a haploid plentiful gene.

[0131] For example, the second construct can comprise a nucleotide sequence encoding a gRNA that, in the presence of the Cas9 nuclease encoded by the first construct, cuts the wild-type allele of the disrupted gene, thereby increasing the transmission of the construct to the next generation by homing or by disrupting chromosomes carrying the wild-type allele. Alternatively, the second construct can also encode Cas9 or any other appropriate nuclease. In either case, the second construct temporarily increases the frequency of the first construct, making it more efficient while maintaining localization ( Figure 3 ). Thus, in one embodiment, the second gene construct comprises a nucleotide sequence encoding a guide RNA that targets the integration site of the first gene construct. Thus, the second construct helps to temporarily increase the frequency of the first gene construct.

[0132] Alternatively, the second construct may comprise a first nucleotide sequence and a second nucleotide sequence encoding the first gRNA and the second gRNA, respectively. The first gRNA preferably allows the first construct to home, and the second gRNA preferably allows the second construct to home in the presence of the first construct, resulting in a dual drive that can spread to other populations, and can control its spread by exploiting pre-existing sequence differences between target and non-target populations ( Figure 4 ). Therefore, in another embodiment, the second gene construct preferably comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence encodes a first guide RNA that targets the integration site of the first gene construct, and the second nucleotide sequence encodes a second guide RNA that targets the integration site of the second gene construct. Therefore, when the first gene construct and the second gene construct are inherited together, this facilitates homing and increases their frequency.

[0133] Alternatively, other methods can be used to enhance the first gene construct. For example, in one embodiment, the first gene construct can include a nucleotide sequence encoding a rescue construct that masks the effects of the mutation produced by the second construct. Alternatively, other gene constructs that increase the frequency of the second construct can be used.

[0134] In an eighth aspect, there is provided a genetically modified organism obtained or obtainable by the method of the fifth, sixth or seventh aspect.

[0135] In a ninth aspect, a genetically modified organism is provided, comprising a disrupted haploid-sufficient gene required for the survival or reproduction of a male and / or female organism, and a nucleotide sequence encoding a genome editor that generates a dominant lethal mutation or a sterility mutation in the target gene, rendering the male and / or female organism comprising the dominant lethal mutation or the sterility mutation infertile.

[0136] Preferably, the haploid plentiful gene has been disrupted by a gene construct as defined in the first aspect. Alternatively, the gene construct has been inserted into or near a disrupted allele of the haploid plentiful gene. Preferably, the haploid plentiful gene, target gene, gene construct and organism are as defined in the first aspect.

[0137] In a tenth aspect, a genetically modified organism is provided, which comprises a disrupted haploid sufficient gene required for the survival or reproduction of male and / or female organisms, and a nucleotide sequence encoding a first genome editor and a second genome editor, wherein the first genome editor produces a dominant lethal mutation or a sterility mutation in a target gene expressed in male and / or female organisms, so that a heterozygote of the dominant lethal mutation or the sterility mutation is lethal or sterile, and the second genome editor produces a recessive lethal mutation or a sterility mutation in a haploid sufficient gene expressed in male and / or female organisms, so that a homozygote of the recessive lethal mutation or the sterility mutation is lethal or sterile.

[0138] In an eleventh aspect, a method for suppressing a wild-type population of an organism is provided, the method comprising cultivating a genetically modified organism comprising a genetic construct comprising a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence being configured to disrupt a haploid-sufficient gene required for survival or reproduction of male and / or female organisms, or being configured to integrate into or near a disrupted allele of a haploid-sufficient gene, the second nucleotide sequence encoding a genome editor that produces a dominant lethal mutation or a sterility mutation in a target gene of the male and / or female organism such that: homozygotes for the genetic construct are lethal or sterile, and / or heterozygotes for the dominant lethal mutation or sterility mutation are lethal or sterile.

[0139] In some embodiments, the disrupted allele of a haplosufficient gene comprises a knockout mutation, optionally, the knockout mutation results from the introduction of a premature stop codon, or alternatively, the knockout mutation can result from the partial or complete deletion of a haplosufficient gene.

[0140] In some embodiments, a method of suppressing a wild-type population of an organism is provided, the method comprising breeding a genetically modified organism comprising a genetic construct comprising a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence being configured to disrupt a haplosufficient gene required for survival or reproduction of the male and / or female organism, the second nucleotide sequence encoding a genome editor that generates a dominant lethal mutation or a sterility mutation in a target gene in the male and / or female organism, rendering homozygotes for the genetic construct lethal or sterile.

[0141] In a twelfth aspect, a method for suppressing a wild-type population of an organism is provided, the method comprising cultivating a genetically modified organism comprising a genetic construct comprising a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence being configured to integrate into a position outside a haplosufficient gene required for survival or reproduction of the male and / or female organism, the second nucleotide sequence encoding: (i) a first genome editor, the first genome editor generating a dominant lethal mutation or a sterility mutation in a target gene expressed in the male and / or female organism; and (ii) a second genome editor, the second genome editor generating a recessive lethal mutation or a sterility mutation in a haplosufficient gene expressed in the male and / or female organism, such that homozygotes for the recessive lethal mutation or sterility mutation are lethal or sterile.

[0142] In a thirteenth aspect, there is provided use of the genetic construct of the first or second aspect for suppressing a wild-type population of an organism.

[0143] To suppress a population, modified organisms can be released in a variety of ways. For example, in one embodiment, a modified organism containing a genetic construct is released into a population. Alternatively, in another embodiment, females containing the genetic construct that have been mated with males are released into the population. Alternatively, the genetic construct can be contained in pollen and then released into the population. In other words, organisms at any appropriate stage of their life cycle can be released to suppress wild-type populations.

[0144] In one embodiment, the inhibition can be localized. This means that regardless of gene flow, the construct and the inhibitory effect will not spread indefinitely throughout the species, but will be confined to a region surrounding the release site. This region typically includes an area within 2, 4, 6, 8, or 10 times the average diffusion distance of the target species from the release site.

[0145] In another embodiment, the inhibition can be partial and non-localized. This means that the construct and the inhibitory effect will spread from the release site through successive generations to all areas within the species range, as long as there is significant gene flow, even after only a single release. In this case, the effect is inhibition of at least 20%, 30%, or 40% of the target population, and preferably no more than 70%, 80%, or 90%.

[0146] In another embodiment, the inhibition can be complete and non-localized. This also means that the construct and the inhibitory effect will spread from the release site to all areas within the species range through successive generations, as long as there is significant gene flow, even after only a single release. The effect in this case is that the target population is suppressed by at least 90%, 95%, or 99%, or preferably eradicated (although the location may be recolonized by the target species after eradication).

[0147] Preferably, the haplosufficient gene, target gene, genetic construct and organism are as defined in the first aspect or the second aspect.

[0148] All features described herein (including any accompanying claims, abstract, and drawings) and / or all steps of any disclosed method or process may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] For a better understanding of the present invention and to show how embodiments thereof may be implemented, reference will now be made, by way of example, to the accompanying drawings, in which:

[0150] Figure 1Combinations of fitness parameters suitable for localized population suppression are shown. The proposed construct is inserted and disrupts essential genes, resulting in lethality or sterility. The construct encodes a genome editor that targets a site in the wild-type allele to cause a mutation that results in dominant lethality or sterility. The table shows the combination of fitness effects of gene disruption (x), target site editing (y), heterozygosity of disruption and editing alleles (x / y), and edited expression, which, after repeated release in 5% of male heterozygotes of the initial male population, results in effective localized population suppression. The phenotypic effects of construct insertion or editing can be male-specific (m or M), female-specific (f or F), or bisexual-specific (b or B), where lowercase and uppercase represent recessive and dominant effects, respectively, and the two letters represent different dominant patterns in the two sexes. For single-locus designs, the x / y column records the phenotype of a heterozygous individual carrying one copy of the construct and one edited allele on the homologous chromosome. Editing can be controlled by, for example, expressing Cas9 or gRNA from a specific promoter, resulting in editing in both sexes (B) or only in males (M) or females (F). In the localizable strategy shown, construct insertion and gene disruption result in recessive lethality or sterility in both sexes, and the genome editor can produce dominant bisexual (green) or dominant female-specific (blue) lethal or sterile editing, or female-specific dominant lethal or sterile editing with male-specific recessive lethality (orange). The figure shows a time course simulation of the relative female population size after repeated release of the construct of the present invention compared to the release of sterile males (black). For the purpose of simulation, all fitness and editing parameters are idealized (i.e., 0 or 1).

[0151] Figure 2 The molecular configurations proposed for achieving localized population suppression according to various embodiments of the constructs of the present invention are shown. In (a), the construct (here exemplified by Cas9 and gRNA) is inserted into a haploid-sufficient essential gene by homologous recombination, which may induce a dominant negative editing (HS ETDN =( ... ETDNGene, resulting in recessive lethality or infertility (for example, by introducing premature stop codons). The construct encodes a genome editor (such as Cas9 and gRNA, although other genome editing tools such as Cas9 derivatives, TALEN or zinc finger nucleases can be used), which targets the site in the wild-type allele of the same gene downstream of the insertion site, resulting in dominant negative editing, which results in dominant lethality or infertility when there is no insertion on the chromosome. The presence of the construct prevents dominant negative from being expressed, so individuals that inherit a copy of the construct and a wild-type allele have a fitness close to normal (although if homozygous for the construct, they will be non-viable or infertile). If the editor works by producing double-strand breaks, it should not cause the construct to home, because the inhibition would not be localized. The insertion site of the construct is shown as upstream of the target site, but it can also be located downstream, in which case the target site on the chromosome containing the insertion must be recoded or removed so that the chromosome carrying the construct is not recognized by the editor. More generally, the chromosome containing the construct can be missing some or all of the wild-type HS ETDN Gene. Genome editors can be designed to produce dominant negative mutations that affect both males and females (left), or to target and edit sequences in female-specific exons to produce dominant negative mutations that affect only females (right). The latter approach can be achieved by using genes such as doublesex. In (b), the construct still inserts and destroys haplosufficient (HS) essential genes, and targets very tightly linked genes to cause dominant lethal mutations or sterility mutations. The target gene can be a haploinsufficient (HI) gene and edited for knockout, or it can be any other gene that can undergo dominant lethal mutations or sterility mutations. The dominant mutations produced by the editor can be lethal or sterile in males and females, or lethal or sterile only in females. The copy of the gene on the same chromosome as the construct has been modified to make it resistant to the editor by changing the target sequence, so it is still functional but is no longer recognized by the genome editor, as shown on the left side of the figure (rHI represents recoded haploinsufficient). However, if the editor produces a dominant negative mutation (Gene ETDN), then the resistant version may be only a deletion of the target site or even a deletion of the entire gene, as shown in the right side of the figure. In (c), the construct targeting can be edited as a dominant negative gene or a haploinsufficient (HI) gene, which can be linked to the construct at a longer distance (including on different chromosomes), and the construct also contains a recoded rescue copy of the target gene that is not recognized by the editor. In the case where the target gene is HI, the rescue gene will be a modified version of the target gene, which still has function, but is not recognized by the genome editor. If the editing produces a dominant negative mutation, the rescue gene is still a modified version of the target gene, which still has function, but is not recognized by the genome editor, and can be designed to have a higher expression level, to more effectively dilute the dominant negative mutant protein. Similarly, the dominant mutation produced by the editor can be lethal or infertile in males and females, or only lethal or infertile in females. For illustration, the gene constructs in Figure ac are shown as inserting and destroying haplosufficient genes. However, the genetic construct can also be configured to be integrated into the disrupted allele of a haploid sufficient gene, where the disruption can be due to the introduction of a premature stop codon or to all or part of the gene being deleted (d; left and center). Alternatively, the genetic construct can be integrated into a position outside but near the disrupted allele (d; right).

[0152] Figure 3 (a) shows the proposed molecular configuration, where Figure 1 The embodiment is paired with a second construct comprising a gRNA - the gRNA is inserted into the neutral (Ntrl) locus - targeting the locus of insertion of the first construct, contributing to its temporary increase in frequency by homing. For illustration, the frequency of the first construct at a single locus (e.g., Figure 2 a) implemented the bBBB design (from Figure 1 ), where the gRNA from the second construct binds to the Cas9 from the first construct, cleaving the wild-type allele at the insertion site of the first construct. Alternatively, the second construct can encode its own Cas9 or other RNA-guided nuclease to allow homing of the first construct. (b) Time course simulation showing the allele frequencies of the proposed constructs (solid line) and the effect on relative population size (dashed line) when a single release of the two constructs in the same males is made at 30% of the initial male population size. For the purposes of this simulation, all fitness and cleavage parameters are idealized (0 or 1) and the constructs are assumed to be unlinked. Note that the enhancer gRNA can be used with Figure 2 When used together with any of the molecular configurations shown in , it has a similar effect of increasing the amount of release.

[0153] Figure 4 Shows (a) the use of Figure 1For illustration, the molecular configuration of the construct bBBB is as follows: Figure 2 a shown on the left, but this strategy applies to Figure 2 . Two constructs are shown, the α construct and the β construct. The β construct contains two gRNAs, one targeting the insertion site of the α construct and the other targeting its own insertion site, both of which promote homing by using the Cas9 encoded by the α construct, resulting in both constructs being able to spread from a low frequency state. If the β construct is designed to insert into a differentiated site (where there is a degree of resistance to the gRNA), localization can be achieved as long as the frequency of resistance in the target population is low enough to allow the spread of both constructs, and the frequency of resistance in the non-target population is high enough to prevent the spread of the β construct and produce the same Figure 3 (b) Time course simulation, showing the allele frequency (solid line) and the effect (dashed line) of the proposed construct when the proposed construct in the same male is released into the population (with 0% and 100% resistance at the differentiation insertion site of the β construct) at 1% of the initial male population size. The figure at the bottom shows the simulation results of 100% resistance on a logarithmic (log) scale to more clearly show the dynamics of the second construct. For the population with 0% resistance, the allele frequency of the two constructs is the same. For the purpose of simulation, all parameters fitness and cutting parameters are idealized (0 or 1), and it is assumed that the construct is non-linked.

[0154] Figure 5 The table displayed shows the combination of gene disruption (x), target site editing (y), heterozygosity of disruption and editing alleles (x / y), and fitness effects of editor expression, forming a self-sustaining population suppression strategy that can suppress more than 99% of the population after a single release of a single-locus molecularly designed construct or a construct with two closely linked loci. The phenotypic effects of construct insertion or dominant-negative editing can be male-specific (m or M), female-specific (f or F), or bi-sex specific (b or B), where lowercase and uppercase indicate recessive and dominant effects, respectively, and the two letters indicate different dominance patterns in the two sexes. The x / y column records the phenotype of a heterozygous individual carrying one copy of the construct and one editing allele on the homologous chromosome. Editing can be controlled by, for example, expressing Cas9 or gRNA from a specific promoter, resulting in editing in both sexes (B) or only in males (M) or females (F). (b) Time course simulation of relative female population size following the design described in the single release table (when released in heterozygous males at 50% of the initial male population size). For the purposes of the simulations, all fitness and editing parameters were idealized (0 or 1).

[0155] Figure 6 Examples of single-locus (a, b) and double-linked loci (c, d) molecular configurations that produce complete suppression in closed, randomly mating populations are shown. In each case, if the terms "female" and "male" are interchanged, the strategy generally still works, although with slightly different kinetics. The configuration with two linked loci has a haplosufficient gene (into which the construct is inserted) coupled to a haploinsufficient (HI) gene or a haplosufficient (HS) gene that can be edited to be dominant negative. ETDN) genes are closely linked, and modification involves insertion of the construct and recoding of the target site so that it is functional but not recognized by the editor. (a) Left: The construct is inserted into and disrupts a haploid-sufficient gene required only for females, with the resulting edit being a female-specific dominant-negative or dominant-gain-of-function mutation. Right: Alternatively, the construct is inserted into and disrupts a female-specific exon of a gene required for both sexes, with the resulting edit being a female-specific dominant-negative. In both cases, the insertion site of the construct is shown as upstream of the site targeted by the editor, but the approach can also be applied if the editor's insertion site is downstream of the site targeted by the editor, although in this case the target site on the chromosome containing the construct needs to be modified or removed so that the chromosome containing the construct is not recognized by the editor. (b) The construct is inserted into a haploid-sufficient gene required for both males and females, but either inserted into a female-specific exon (left) or with a sequence at its end that ensures it will be spliced ​​out in males (right), resulting in gene function being disrupted primarily in females. The editing produced in this way is dominant negative for both sexes, and because the target site has been recoded, the chromosome where the construct is located is protected from the effects of editing. (c) The construct is inserted into and destroys the haploid sufficient gene required for females (left side), or is inserted into and destroys the haploid sufficient gene required for both sexes and has a sequence at its end that ensures that it will be cut out in males (right side), resulting in gene function being destroyed mainly in females. The genome editor acts on the wild-type allele located in a gene that is different from the destroyed gene and is tightly linked, producing a dominant negative mutation, a dominant gain-of-function mutation, or a knockout of a haploinsufficient gene. In both cases, because the target site has been recoded, the chromosome where the construct is located is protected from the effects of editing. (d) As shown in (c), the difference is that the construct destroys the haploid sufficient gene required for both males and females, and the editor targets the tightly linked gene required for both sexes. Left side: The editor produces a dominant negative mutation, and the target gene on the chromosome containing the construct has been modified so that it is not recognized by the editor but still has function. In addition, the control sequences have been modified to specifically increase expression in males, thereby diluting the effect of the dominant negative edit in males heterozygous for the construct and the edit, resulting in only female heterozygotes being lethal or sterile. Right: The editor creates a knockout mutation in the haploinsufficient gene, and the target gene on the chromosome containing the construct has been modified so that it is not recognized by the editor but remains functional. In addition, a second copy of the gene, also recoded so as not to be recognized by the editor, has been inserted into control sequences that limit male-specific expression, resulting in: organisms heterozygous for the construct and the edit have normal or near-normal fitness if they are males, but are sterile or lethal if they are females.Male-specific expression can be achieved by modifying promoter or enhancer sequences, or by using introns that are spliced ​​out only in males, or by introducing premature stop codons if splicing is not performed.

[0156] Figure 7 Shown are (a) a table showing the combinations of gene disruption (x), target site editing (y), and fitness effects of edit expression that form a self-sustaining population suppression strategy capable of suppressing the population by over 99% after a single release of a construct with a two-locus molecular design. (b) a time course simulation of the relative female population size following a single release of the design described in the table (when released in heterozygous males at 50% of the initial male population size). For the purposes of the simulations, all fitness and editing parameters were idealized (0 or 1) and the two loci were assumed to be unlinked. Labels such as Figure 5 shown.

[0157] Figure 8 Included are tables showing the combinations of gene disruption (x), target site editing (y), heterozygosity for both disrupted and edited alleles (x / y), and fitness effects of edit expression that, in our simulations, result in self-sustaining population suppression strategies capable of partial suppression of the population by 50% to 99% after a single release of a construct with a single-locus molecular design or a construct with two closely linked loci. All labels in the table are the same as Figure 5 The figures show the time course simulations of the relative female population size after a single release of the designs described in the table, when released in heterozygous males at 50% of the initial male population size, with (a) ideal parameters, or (b) non-ideal parameters, where the fitness cost of recessive heterozygotes is 5% and the editing efficiency is 80%. The black line is a construct with a bisexual recessive phenotype, the pink shading shows a construct with a female-specific recessive phenotype, and the blue is a construct with a male-specific recessive phenotype. (c) and (d) show the time course simulations of the six most efficient strategies (measured by the time required to reach equilibrium) when two additional releases are performed, including more than one construct of the same design located elsewhere in the genome. In the second release at generation 35, males carry one copy of the first construct and one copy of the second construct. In the third release at generation 70, males contain one copy of each of the three constructs. (c) shows the results for the ideal parameter values, and (d) shows the results for the non-ideal parameter values.

[0158] Figure 9Included are (a) tables showing the combinations of gene disruption (x), target site editing (y), and fitness effects of edit expression that, in our simulations, form self-sustaining population suppression strategies capable of partial suppression of the population by 50% to 99% after a single release of constructs with a two-locus molecular design. Figure 5 Figures (b and c) show the time course simulation of the relative female population size after a single release of the design described in the table when released in heterozygous males at 50% of the initial male population size, where (b) is the ideal parameter, or (c) is the non-ideal parameter, where the fitness cost of recessive heterozygotes is 5% and the editing efficiency is 80%. In each case, the two loci are unlinked.

[0159] Figure 10 Examples of single locus (ad), double linked locus (eg), and double unlinked locus (h, i) molecular configurations are shown, resulting in partial suppression in closed, randomly mating populations. (a) The construct is inserted and disrupts a haploid sufficient gene required for both males and females, resulting in a bisexual dominant negative edit. In an organism heterozygous for the wild-type allele of the genome editor and the HS gene, the editor acts on the wild-type target sequence on the chromosome that does not carry the construct, generating a dominant negative mutation that is lethal or sterile for both males and females; the target site on the chromosome carrying the construct has been recoded so that it is still functional but cannot be recognized by the editor (rHS ETDN; no orange arrows). The construct can also provide dominant protection against the mutation by nullifying the effects of the mutation at the RNA level, for example by degrading RNA transcribed from the mutated target gene through RNA interference, or by restoring RNA transcribed from the mutated target gene to the wild-type sequence through RNA editing. Note that if the construct encodes an RNA editing module, there is no need to recode the target site on the chromosome carrying the construct, because the RNA transcribed from it will also be restored to wild type. (b) Left: The construct is inserted into and disrupts a haploid-abundant gene required for females, and the resulting edit is a female-specific dominant negative. Right: Alternatively, the construct is inserted into and disrupts a female-specific exon required for both sexes, and the resulting edit is a female-specific dominant negative. (c) The construct is inserted into a haploid-abundant gene required for both males and females, but either inserts a female-specific exon (left) or has a sequence at its end that ensures it will be spliced ​​out in males (right), resulting in gene function being disrupted primarily in females. The resulting edit is a bisexual dominant negative. In (b) and (c), the construct also invalidates the mutation effect at the RNA level by encoding an RNAi module or an RNA editor, and the target site on the chromosome carrying the construct has been recoded so that it still has function but is not recognized by the editor. (d) The construct is inserted into the haploid sufficient gene required for both males and females, but inserted into the male-specific exon, resulting in gene function being destroyed mainly in males. The editing produced in this way is a female-specific dominant negative, which is achieved by targeting the female-specific exon of the same gene, and because the target site has been recoded, the chromosome where the construct is located is protected from the influence of the editor. (e) The construct is inserted into and destroys the haploid sufficient gene required for females (left side), or inserts and destroys the haploid sufficient gene required for both sexes, with a sequence at its end that ensures that it will be cut out in males (right side), resulting in gene function being destroyed mainly in females. The genome editor acts on the wild-type allele in a gene that is different from the destroyed gene and is tightly linked, producing a dominant negative mutation, a gain-of-function mutation, or a knockout of a haploinsufficient gene. (f) As in (e), where the target gene is female-specific. Note that in both (e) and (f), the target site on the chromosome carrying the construct has been recoded so that it is still functional but cannot be recognized by the editor, and the construct can also invalidate the mutation effect at the RNA level (i.e., by encoding an RNAi module or RNA editor). (g) The construct is inserted into and disrupts a haploid gene required for females (left), or inserted into and disrupts a haploid gene required for both sexes, and its end has a sequence that ensures it will be cut out in males (right), resulting in gene function being destroyed primarily in females.Genome editors act on the wild-type allele in a gene that is different from and closely linked to the gene being disrupted, resulting in a dominant negative, a gain-of-function mutation, or a knockout of a haploinsufficient gene, any of which results in lethality or sterility in males. Note that in the cases of single-locus (d) and two-locus linkage (g), the construct does not need to provide dominant protection against editing if the sex affected by the disruption is the opposite of the sex affected by the edit. (h) Constructs are inserted and disrupt a haplosufficient gene required for females (left), or a haplosufficient gene required for both sexes with sequences at its ends that ensure it will be spliced ​​out in males (right), resulting in disruption of gene function primarily in females. Genome editors act on the wild-type allele in a gene located distant from the disrupted gene, resulting in a dominant negative, a gain-of-function mutation, or a knockout of a haploinsufficient gene, any of which results in lethality or sterility in males. In cases where editing is performed in a gene unlinked to the disruption, the construct must also encode a recoded version of the target gene that is resistant to editing while also restoring function in individuals carrying a single copy of the dominant edit. (i) As shown in (h), where the editing results in female-specific (rather than male-specific) dominant sterility or lethality, and the editing occurs only in males (e.g., Cas9 expression is controlled by a promoter that is active only in males). Note that in d, g, and h, if the terms "female" and "male" are interchanged, the strategy generally still works, although with slightly different kinetics.

[0160] Figure 11 The molecular configurations of various embodiments of the constructs of the present invention are shown, providing localized population inhibition, non-localized partial inhibition, or non-localized complete inhibition, wherein a second gRNA in the construct is used to induce the recessive effect of the construct. a) shows the use of a single HS ETDN The molecular mechanism of genes (similar to Figure 2 a Design on the left). Here, the construct encodes a genome editor that i) targets HS ETDN) the wild-type allele of the gene, resulting in dominant lethality or infertility of both sexes; ii) targeting the allele linked to the construct and located upstream of the dominant mutation to produce an early termination codon, thereby resulting in recessive infertility or lethality of both sexes. The presence of an early termination codon prevents the expression of a dominant negative mutation downstream, so that an individual inheriting a copy of the construct and the termination codon associated therewith and a wild-type allele has a fitness close to normal (although if the construct and the homozygote of the termination codon associated therewith are, they are non-viable or infertile). By recoding the allele linked to the construct, it is still functional but can be targeted by a genome editor, while the wild-type allele is unaffected, and specificity for the allele linked to the construct can be achieved. The gene construct can also be configured to induce recessive infertility or lethal mutations in the gene, which is independent of the gene targeted by the first gRNA to induce dominant infertility or lethal mutations. In this case, the construct can be linked to the HI gene or any gene targeted by the first gRNA that may produce a dominant lethal mutation or sterility mutation in both sexes or only in females (similar to Figure 2 The construct in b, such as Figure 11 b) or unlinked (similar to Figure 2 c constructs, such as Figure 11 c), and in both cases, the construct can be linked or unlinked to the HS gene targeted by the second gRNA to induce a recessive lethal mutation or sterility mutation. DETAILED DESCRIPTION

[0161] Example

[0162] The inventors sought to identify a genetic construct that could be inserted into a species and released into a population to effectively suppress the population over a period of time. Therefore, the inventors developed a novel genetic construct that could improve the efficiency of population suppression due to its ability to persist in the population for multiple generations. The inventors discovered that, depending on the precise configuration, the genetic construct, when released into the target population, could produce three different types of potentially useful effects: (i) localized suppression, (ii) non-localized partial suppression, or (iii) non-localized complete suppression.

[0163] Materials and methods

[0164] Model

[0165] 1. Overview

[0166] The following sections will illustrate the model for simulating the suppression strategy. The model follows the deterministic structure developed by Burt and Deredec (2018, P.Roy.Soc.Biol.Sci.285 (1883)) to simulate a single, randomly mated population of infinite size with discrete, non-overlapping generations and two sexes. The model is programmed using the scientific programming language Julia (Bezanson et al., 2012, arXiv:12095142), and all simulations are performed in the Jupyter Notebook interface (Kluyver et al., 2016, IOS Press). For basic single construct design, the inventors simulated the release of a single gene construct, which is edited at a specific site in the genome, and explored two situations. The first situation is that the target site is located in the same locus as the construct, and the second situation is that the target site is located at other positions in the genome. For the localized suppression strategy, the inventors also simulated a second construct, which allows the first construct to return to its nest, and allows potential itself to return to its nest. The inventors also simulated the release of multiple constructs, each with the same design as a simple single construct, where each construct was located in and targeted a different locus than the first construct.

[0167] To simulate each situation, the inventors used a flexible simulator that can simulate multiple loci (n=L), each of which can contain several alleles, one of which can contain a construct. The inventors simulated the population by tracking all possible individual genotypes (including all possible allele combinations in all considered loci) and allowed the linkage between each pair of loci to change. Depending on the design of the construct, it can affect gamete transmission by cutting the wild-type (WT) allele of any simulated locus, causing the allele to home to the homologous chromosome, or by mutating the wild-type allele. By changing the genotype-specific fitness cost associated with host gene destruction and editor-induced mutations, the inventors allowed the construct to be inserted into a variety of different genes (such as haplosufficient genes, haploinsufficient genes, etc.). The following sections will illustrate how to implement these processes in the model.

[0168] 2. Gamete transmission

[0169] Depending on the genotype, the probability of gametic transmission may be altered due to cleavage at the target site. This is simulated using a set of locus (L) and sex (G) specific parameters. Cleavage requires the presence of one or more constructs that together contain components capable of site-specific cleavage (e.g., Cas9 and gRNA) and the corresponding target allele, assuming that the activity associated with each construct component is dose-independent. In sex G, the probability of cleavage of the WT at locus L is In heterozygotes at the target site, repair of the cut chromosome can occur by nonhomologous end joining (NHEJ) with a probability of The WT was converted into the cleavage-resistant mutant alleles r1 and r2 with the probabilities of and Alternatively, repair can occur via homology-directed repair (HDR), where homing occurs with a probability of In the case where cleavage occurs at a locus homozygous for the WT allele, the probability of cleavage and subsequent mutation occurring for each WT allele is Thus, WT is converted to r1 or r2 alleles in a ratio of When simulating more than one locus, we allow recombination to occur between each pair of loci during gamete production. Assuming the loci are arranged linearly along the chromosome (from A to Z), r i Describes the probability of recombination between loci i and i+1, requiring a total of r L-1 Parameters, where L is the total number of simulated loci. Assuming that the recombination occurring at different positions on the chromosome is independent (i.e., there is no interference), r = 0.5 can be used to simulate loci on different chromosomes. For reference, a record of all the parameters included in the simulator can be found in Table 1.

[0170] 3. Fitness Effect

[0171] The inventors allow the destruction and / or editing of host genes to affect the fitness of the genotype relative to WT. In order to simulate these fitness effects, for simplicity, the inventors set two different fitness spectra for each locus, wherein the first fitness spectrum describes the fitness caused by the insertion of the construct or the r2 mutation (I), and the second fitness spectrum describes the fitness associated with the r1 mutant (R). The inventors applied this principle to all loci, using a set of locus-specific and sex-specific parameters to simulate the fitness spectrum of each locus. If W is the WT host gene, D is the allele containing the construct, and r2 and r1 are anti-cutting mutant alleles, then due to the destruction of the host gene at the L locus, the fitness of each individual relative to WT is:

[0172]

[0173] or

[0174] or

[0175]

[0176] in, and is the selection coefficient, and are dominance coefficients, each of which varies by locus and sex.

[0177] If more than one construct present in the genome carries a rescue component specific for the edited gene, the fitness cost caused by editing can be reduced. To simulate this, the inventors calculated the number of functional copies (including WT and rescue copies) of locus L present in the genotype. When there are two functional alleles, the relative fitness caused by host gene disruption is 1. When there is a single functional allele, the relative fitness caused by host gene disruption is Here, the inventors assumed that the WT and rescue copies were functionally equivalent and that there was no additional cost when more than two functional copies of a gene were present.

[0178] 4. Population biology

[0179] The model simulates the evolution of a population over time, assuming discrete, non-overlapping generations and two life stages (larvae and adults), where larval survival is density-dependent according to the Beverton-Holt model. In each generation, adult males and females produce gametes, during which recombination occurs between the two loci, and homing or mutation may occur in males or females, depending on the genotype. The population is assumed to mate randomly, with females producing f(N m / (N m +bN eq )) fertilized eggs. When b is not equal to 0, the number of eggs allowed to be laid depends on the number of males present (N m ). The survival rate of the larvae is density-dependent, so that the survival probability is where q J is the density-independent probability that a juvenile will survive to adulthood, a determines the magnitude of density-dependent mortality, and Z is the number of juveniles in the population. All results are reported here for relative population size (compared to the equilibrium state before release) and are therefore not affected by the exact value of a. When b = 0, the intrinsic growth rate (R) of the wild-type population is m )for For simplicity, all genotype-dependent fitness costs associated with host gene disruption or construct activity were assumed to affect survival after density-dependent larval mortality and before population surveys (e.g., in the case of pupal mortality).

[0180] Table 1 - Model parameters

[0181]

[0182] L represents the locus and G represents the sex of the individual.

[0183] filter

[0184] The inventors first simulated the release of multiple different constructs based on a single locus design, where the construct could cause a phenotypic effect by disrupting the function of its insertion site and generating a mutation at that location within the same gene. Here, the inventors simulated a single autosomal locus with three alleles: a wild-type allele, a transgenic construct that inserted and disrupted the wild-type allele, and a non-functional mutation generated by the construct. The inventors simulated situations where a construct could be inserted into a gene, resulting in a dominant adult. or implicit Lethal or neutral The gene can be sex-specific, with G=F in females and G=M in males. Similarly, the inventors allow the phenotypic effect of the mutation to be dominant in either sex or both sexes. Hidden or neutral The inventors also allowed for a neutral fitness effect on individuals heterozygous for the construct and the mutant allele. or cause death / infertility Still allowing these effects to be different in different sexes. Finally, the inventors allowed sex-specific mutations based on the expression activity of the construct, where mutations could occur only in males (c m =1,c F =0) or female (c m =0,c F =1), or both sexes (c m =1,c F =1). All end connection rates (j G ) were all equal to 1 to ensure that all cut alleles resulted in mutations and that homing of the construct was impossible. They also set p G = 1 to ensure that all mutations generated by the construct are of the same type in terms of fitness effect and their fitness can be different from that of the construct itself.

[0185] The inventors performed similar simulations, assuming that the edits produced by the construct were located elsewhere in the genome and that the construct contained a recoded version of the target locus. Here, the inventors simulated two loci, each with two alleles, and assumed that the two loci were unlinked (r=0.5). At the first locus (A), the two alleles contained wild type and transgenic constructs; at the second locus (B), the inventors included wild type and mutants produced by the construct. Similarly, the inventors allowed the fitness effect of the construct to vary, resulting in adults being dominant. or implicit Lethal or neutral and sex-specific, with G=F for females and G=M for males. Similarly, the inventors allowed the phenotypic effect of a mutation at locus B to be dominant in either sex or both sexes. or neutral Finally, the inventors have allowed sex-specific cleavage at the B locus based on the expression activity of the construct, where cleavage can occur only in males. or female or both sexes The inventors assumed that the probability of cleavage of the WT allele in heterozygotes and homozygotes is the same. The inventors also hypothesized that the recoded version of the target locus is functionally equivalent to the WT allele, i.e., in the presence of two editing alleles, one copy of the construct is sufficient to rescue edits with a recessive phenotype, but not sufficient to rescue edits with a dominant phenotype.

[0186] For all possible combinations of these parameter groups for two different designs, the inventors simulated a single release of male heterozygotes at a ratio of 50% of the initial male population and monitored the relative number of females for 200 generations. The inventors divided the results into three groups based on their kinetics. The performance of strategies with suppression below 50% in repeated releases was studied, and a release of 5% per generation was simulated. Those with relative female population sizes <1% were further studied for complete eradication; those with relative female population sizes between 1% and 50% were further studied for partial suppression. In the last group, the inventors repeated the simulations using less than ideal parameters to determine the strategy that was most robust to fluctuations in fitness and cutting parameter values ​​(when the construct or target site mutant was a recessive mutation, the fitness cost of the heterozygote was 5% and the cutting efficiency was 80%).

[0187] Identify target genes

[0188] A reasonable course of action for implementing the present invention for localized inhibition would be to first sequence the genome of the target species (if it does not already exist) and then search for homologs of known genes with the desired properties.

[0189] For example, homologues of the doublesex gene are suitable.

[0190] In addition, any gene that is haplosufficient and editable to produce a dominant sterile or lethal phenotype can be used. A FlyBase search revealed that 45 genes have both dominant and recessive lethal mutations, including 5-HT2A, Antp, BicD, cact, chic, Col4a1, crn, cype, dare, dl, dpp, Fs(2)Ket, gro, hb, hh, hop, Hsc70-3, Hsc70-4, ken, l(1)10Ad, l(1)10Ae, l(2 )25Ca, l(2)40Ff, l(2)46Fb, l(2)DTS18SP, l(2)DTS19, l(2)DTS20, l(2)DTS6, l(2)DTS8, l(2) DTS9, l(2)M167, lt, M(3)80, nos, Prosbeta6, puc, rl, Scr, snf, stmA, Sxl, tkv, Tl, tor, wupA.

[0191] To confirm a recessive phenotype, one can experimentally knock out the target gene in the target organism to confirm the recessive phenotype (bisexual lethality or sterility) and generate truncating mutations (by introducing a premature stop codon) in the last two exons, the last three genes, a certain number of bp from the end, or other edits suggested by homology, and confirm the dominant phenotype (female or bisexual lethality or sterility).

[0192] If both phenotypes are confirmed, a haplotype of the invention carrying the disrupted or deleted haplosufficient gene and the editor can be generated, and the haplotype can be ensured that the haplotype is not edited (e.g., because the recognition site for the editor is not present) or the edit is not expressed (e.g., because it occurs after a stop codon).

[0193] Alternatively, if the first experiment confirms the dominant phenotype of the editor, but does not confirm the recessive phenotype of the knockout, nearby genes can be checked to see if there are genes homologous to haplosufficient essential genes in Drosophila (or other model organisms), and then knocked out to confirm the recessive lethal or sterile phenotype. If this is confirmed, a haplotype carrying a disrupted haplosufficient gene, an editor, and a target gene that has been recoded (or deleted) can be produced so that it is not recognized by the editor. In this case, it may be necessary to select an editor that does not stimulate recombination repair, such as a base editor or a reverse leader editor, or an editor that cuts the target site to produce a 3' overhang. In addition, it may be appropriate to reduce the possibility of recombination that destroys the haplotype (for example, by inverting part or all of the sequence).

[0194] Alternatively, if the first experiment confirms the dominant phenotype of the edit, but there are no haplosufficient essential genes nearby, you can search for homologs of known essential haplosufficient genes farther away in the genomic sequence (for example, there are 818 genes in FlyBase that are annotated as having null or loss-of-function alleles, resulting in recessive lethal phenotypes). Select such a gene, confirm that the knockout has a recessive sterility or lethal phenotype, and then construct a haplotype of the invention consisting of the disrupted gene, the editor, and a rescue gene that inhibits the dominant edit (for example, by encoding more than one copy of the target gene, recoding it so that it is not recognized by the editor, or by encoding a function that disables the edit at the RNA level, such as by RNAi or RNA editing).

[0195] Alternatively, if no dominant mutations are found in the homologs of the above-mentioned haplosufficient genes, homologs of known haploinsufficient genes can be sought. Cook et al. (2012: Table 2) listed 43 genes that are haplolethal or haploinfertile in Drosophila melanogaster, and another 6 regions in the genome that appear to have genes that are haplolethal or haploinfertile, but the specific genes have not yet been discovered. Most of these genes encode protein components of the cytoplasmic ribosome (Rp genes) or translation initiation factors (eIF genes) and are associated with Minute syndrome (a set of phenotypic characteristics including short and thin bristles and developmental delay). Therefore, the editor of a preferred embodiment of the present invention targets a homolog of one of these genes, or targets a homolog of another cytoplasmic ribosomal protein or translation initiation factor.

[0196] Other classes of genes that exhibit haploinsufficiency (which would be lethal in nature if not under laboratory conditions) include genes encoding the muscle components actin (Act88F), myosin (Mhc and Mlc2), and tropomyosin (Tm2), as well as a group of tightly linked muscle-related genes regulated by a haplolethal sequence in the intron of the troponin I (wupA) gene. This subset may also include Hdl, which may correspond to troponin T (up). Similar to ribosomal protein genes, these genes may be particularly sensitive to dosage because muscle assembly requires minimum levels or specific stoichiometric amounts of component proteins. Other definable subsets encode homeodomain proteins (Abd-B, Dll, Scr, and Ubx), Notch pathway components (Dl, H, and N), Polycomb family repressors (Pc and Pcl), apoptosis regulators (lok and p53), and melanin biosynthesis enzymes (b and e).

[0197] Then, it can be confirmed experimentally that knockout of the homolog produces a dominant sterile or lethal phenotype in one or both sexes (e.g., by generating premature stop codons). The above method is then continued, first searching for nearby haploid sufficient genes, or searching for haploid sufficient genes further away, and constructing the haplotypes of the present invention accordingly.

[0198] If the goal is to achieve partial or complete control by self-spreading interventions, then the combination of fitness effects of gene disruption will be different, but similar methods using homology and experimentation can be used to construct the haplotypes of the present invention. For example, if the goal is to achieve complete suppression by self-spreading interventions, then one combination to achieve this goal is to have the disrupted haplosufficient gene have a sterility or lethal phenotype only in females, and to have the edit have a dominant effect only in females ( Figure 5 Likewise, homologs of doublesex genes could be suitable targets if recessive disruption and dominant editing are restricted to female-specific exons ( Figure 6 Alternatively, homologs of Drosophila genes that are female-specific haplosufficient and editable to be dominant female-specific negative (including homologs of the Drosophila genes ovo, dorsal, torso, easter, or Toll) may also be suitable ( Figure 6 a Left side).

[0199] If the goal is to achieve partial inhibition through self-diffusion interference, the construct can also encode functions that disable trans-editing at the RNA level, such as through RNAi or RNA editing.

[0200] In some embodiments, the disrupted gene, while essential in nature, is not essential in the laboratory for the purpose of producing homozygous, pure-breeding lines. Thus, the gene disruption may be auxotrophic and can be compensated for by dietary supplementation. The gene may be involved in the biosynthesis of purines, pyrimidines, or fatty acids, or may be rescued by dietary supplementation with these factors, fructose, or linolenic acid. The target gene may be a homolog of the Drosophila genes rudimentary, rudimentary-like, Dhod, ade2, ade3, ade4, ade5, bur, Pgd, or SREBP.

[0201] If the target species is a mammal, those skilled in the art can use a similar approach, using publicly available data (e.g., MGI or OMIM databases) to identify suitable genes as query content, to find homologs in the target species. The mouse genes that may be haploinsufficient or loss of function intolerance have been listed (e.g., https: / / search.clinicalgenome.org / kb / curations), and the human genes (Coban-Akdemir et al. .2018Am J Hum Genet 103:171-187) that can mutate into dominant negative or dominant gain of function alleles. This reference emphasizes that many mutations that produce dominant negative or dominant gain of function alleles are premature termination codons that are located at positions where nonsense-mediated decay does not occur in transcripts.

[0202] result

[0203] Example 1 - Localized Inhibition

[0204] 1.1 Single-locus and double-locus screening

[0205] Single-locus screens identified three strategies that achieved efficient localized suppression with a 5% repetitive release rate over 50 generations, two of which also emerged in the two-locus screen. Figure 1 As shown, two-locus screening is more restricted in the allowed parameter combinations than single-locus screening, and the third strategy is not included in the set of strategies allowed for two-locus screening. Figure 1 It also shows the expected reduction in population size using these strategies compared to releasing sterile males (a closely comparable method widely used to control certain pests) under certain idealized conditions. It can be seen that the efficiency of the constructs of the present invention is significantly higher.

[0206] These strategies involve releasing constructs that cause or are associated with recessive lethal or sterile phenotypes in males and females. The constructs produce bisexual or female-specific dominant mutations, or (in the case of a single locus) produce a female-specific dominant phenotype with a male-specific recessive phenotype.

[0207] 1.2 Molecular configuration

[0208] Figure 2 Three alternative molecular configurations applicable to all three strategies are shown, where editing can occur in (a) the same gene, (b) a different gene tightly linked to the construct, or (c) an unlinked gene, each of which produces the same Figure 1 The construct comprises a nucleic acid sequence encoding a genome editor, which can be any of the following: a transcription activator-like effector nuclease (TALEN) genome editor, a zinc finger nuclease (ZFN) genome editor, or a CRISPR-based genome editor, such as CRISPR-Cas9 used with one or more guide RNA (gRNA) sequences. For illustration, Cas9-gRNA is shown in the figure, but those skilled in the art will appreciate that any other genome editing technology can be used to apply the present invention.

[0209] The sequence encoding the genome editor is flanked by nucleic acid sequences that allow homologous recombination and thus integration into a haploid sufficient gene (HS). Thus, the construct can be inserted into the genome in a manner that disrupts haploid sufficient genes required for male and female survival or reproduction, such as Figure 2 As shown in ac.

[0210] Alternatively, it can be associated with a mutation that causes a recessive lethal or sterility mutation, yet has little effect on fitness when heterozygous (having one wild-type allele). For example, the invention may involve replacing a native gene in a target organism with a gene that has introduced a recessive lethal or sterility mutation, which also contains a genome editor (natural or synthetic) in an intron. Alternatively, the editor can be tightly linked to the gene that has introduced the recessive mutation.

[0211] In summary, localized suppression design must have the following three characteristics:

[0212] a) Gene constructs cause recessive sterility or lethality in both sexes.

[0213] b) Genetic constructs produce mutations that cause dominant sterility or lethality in both sexes or in females only.

[0214] c) The gene construct provides protection against a single copy of the dominant mutation it generates.

[0215] Figure 2 Examples of molecular configurations that can be used for localized population suppression are shown.

[0216] Figure 2 a The left side shows two chromosomes of a diploid individual, one chromosome (top) carries the haploid sufficient gene required for both sexes (which has been destroyed by the insertion of a genome editor (represented by Cas9 and gRNA boxes)), which introduces a premature stop codon in the HS gene, and the other chromosome (bottom) carries the wild-type allele of the haploid sufficient gene. The arrows indicate that in organisms heterozygous for these two alleles, the editor acts on the target sequence downstream of the editor insertion site on both chromosomes. The dominant mutation generated by the editor causes lethality or sterility in a wild-type genetic background. Due to the presence of the premature stop codon, the mutation on the chromosome carrying the construct is not expressed. Therefore, the HS gene must be a gene that may introduce a dominant negative mutation (DN) or a dominant gain-of-function mutation, so it is labeled HS ETDN (denoting haplosufficient, editable as dominant negative; here and elsewhere, dominant negative and dominant gain-of-function mutations are combined under the label “dominant negative”). Many genes in which loss-of-function mutations are reported as recessive lethal or sterile, and dominant lethal or sterile mutations have been observed, and more generally, both recessive and dominant lethal or sterile mutations have been reported in many genes (see above). Control sequences that determine the tissue specificity of editor expression are selected so that the editor is expressed in the germline so that zygotes heterozygous for the allele carrying the construct and the wild-type allele should have normal or near-normal fitness but pass the edited allele to a large proportion (approximately 50%) of their offspring. The configurations shown are for illustration only and can be modified in many ways without departing from the invention. (1) Multiple variants of the editor are contemplated. The editor can have more than one gRNA (to increase efficiency and / or reduce the likelihood of evolving resistance). Instead of using Cas9, the editor could use a Cas9-derived protein to allow DNA nicking, base editing, prime editing, or other types of editing. Alternatively, it could use Cpf1. Alternatively, it could be based on a TALEN or ZFN-based architecture instead of CRISPR. (2) The insertion site of the construct could be downstream rather than upstream of the target sequence recognized by the editor; in this case, the target site on the chromosome carrying the construct would be modified so that it is not recognized by the editor. (3) The construct is shown inserted into the HS gene, but it could delete part or all of the HS gene (making it more like a gene replacement), or the construct could be inserted outside of but closely linked to the disrupted allele of the HS gene. (4) Optionally, the construct could also encode a marker (e.g., a fluorescent protein) to aid in tracking the construct (not shown).

[0217] Figure 2 a A molecular configuration is shown on the right side, in which a genetic construct (here shown as an example of Cas9 and gRNA) is inserted and disrupted in a haplosufficient gene (HS) required for viability or fertility of both sexes, and the genome editor targets sequences in the female-specific exons of the gene (pink) to produce dominant female-specific lethal mutations or sterility mutations (arrows). The pink and blue thin lines show the sex-specific splicing patterns of the gene; for simplicity, they are shown only on the upper chromosome, but also apply to the lower (wild-type) allele. Homologs of the Drosophila gene doublesex may be genes suitable for this configuration because loss-of-function mutations are recessive infertility in both sexes and they have female-specific exons in which dominant female sterility mutations can be produced. Figure 2 The same changes described on the left side of a apply here (nature of the editor, relative positions of the construct and target site, option to delete many of the HS genes inserted as part of the construct, etc.).

[0218] Figure 2 b shows a molecular configuration in which a gene construct (here shown as an example of Cas9 and gRNA) is inserted to disrupt a haplosufficient gene (HS) required for viability or fertility in both sexes, and the genome editor targets sequences in genes that are distinct from but closely linked to the gene where the gene construct is integrated (left: HI; right: Gene ETDN ). The dominant mutations generated by the editor cause lethality or sterility in a wild-type genetic background. Note that the arrow indicates that the editor only acts to edit the target gene located on the chromosome that does not contain the gene construct. The genome editor can be configured to generate a knockout mutation in the wild-type allele of a haploinsufficient gene (left: HI). In this case, the target site located on the chromosome containing the gene construct contains a recoded version of the target site, making it functional but not recognized by the editor (rHI). Alternatively, the genome editor can be configured to generate a dominant negative mutation in any gene (right; Gene ETDN ), in which case the chromosome containing the gene construct can be rendered resistant to editing by including a deletion of the target site or deletion of the entire target gene (Gene ETDN express). Figure 2 The variants (1), (3) and (4) on the left side of a also apply here (nature of the editor, option to delete many of the HS genes (as part of the construct insertion) and introduction of the marker). For illustration, Figure 2 The left and right sides of b show the editor acting on target sequences in independent genes located downstream of the editor insertion site; however, the target site can also be located in a gene upstream of the editor integration site.

[0219] Figure 2 c shows a molecular configuration in which a gene construct (shown here as an example of Cas9 and gRNA) is inserted into and disrupts a haplosufficient gene (HS) required for viability or fertility in both sexes, and the editor targets sequences in separate genes distant from the gene construct integration site (left; HI, and right; Gene ETDN ). Arrows indicate that in organisms carrying the construct, the editor acts on target sequences on both chromosomes to produce dominant mutations that cause lethality or sterility in the wild-type genetic background. Genome editors can be configured to generate knockout mutations in the wild-type allele of a haploinsufficient gene (left; HI) or dominant negative mutations in any gene (right; Gene ETDN ). In both cases, the construct can also encode a module that provides the organism with protection against one copy of the edited allele (instead of two copies). If the edit is a knockout of a haploinsufficient gene (left), the rescue module can encode a recoded copy of the target gene that the editor cannot recognize and edit, but this recoded copy retains the function of the wild-type target allele. Alternatively, if the edit is a dominant negative mutation, the rescue copy of the construct can be configured to have a higher expression level to dilute the mutation effect. Figure 2 The variants (1), (3) and (4) on the left side of a also apply here (nature of the editor, option to delete many of the HS genes (inserted as part of the construct) and introduction of the marker).

[0220] For illustration purposes, Figure 2 The gene construct in ac was shown to insert into a haplosufficient gene and cause its disruption. Figure 2 The left and center of d show how the constructs can be optionally configured to integrate into a disrupted allele of a haplosufficient gene, where the disruption can be due to the introduction of a premature stop codon (grey) or a complete or partial deletion of the gene (dashed box) (d; left and center). Alternatively, the gene construct can be integrated into a position outside of but near the disrupted allele (d; right). Although all constructs are shown herein as causing disruption (e.g., Figure 2 a), however, all designs can also be constructed so that the editor has no causal relationship with the destruction (as shown in Figure 2 d).

[0221] Recessive lethal or sterility mutations can be selected so that they can be masked in the laboratory and in released individuals, and editors or dominant edits can be designed so that they can be suppressed in the laboratory, thereby producing pure-breeding lines, reducing production costs and allowing the release of homozygous individuals, increasing the efficacy of these releases (or allowing them to be smaller).

[0222] 1.3 Improve efficiency

[0223] The organism carrying the construct can also carry a second construct that causes an increase in the frequency of the first construct. For example, the second construct can encode a gRNA that allows the first construct to home, temporarily increasing its frequency, making it more efficient while maintaining localization ( Figure 3 ).

[0224] refer to Figure 3 a, Shows an example of a molecular configuration for a dual-construct design, where Figure 2 a The gene construct shown on the left (shown here as the right construct) is paired with a second construct (left construct) containing a gRNA – the gRNA is inserted into the neutral locus (Ntrl) – targeting the wild-type sequence inserted by the first construct. Figure 2 As shown in a, the orange arrows show that the editor acts on the target sequence downstream of the editor insertion site on both chromosomes to produce dominant mutations, which result in infertility or lethality in a wild-type background. Alternatively, the target site on the chromosome carrying the construct can be modified independently to produce a dominant negative mutation (not expressed due to the premature stop codon introduced by the construct), and then when the chromosome is cut by the editor, the mutation homes to the chromosome that does not carry the construct. The green arrows indicate that in an organism that is heterozygous for the right construct and carries at least one copy of the left construct, the sequence at the construct insertion site on the wild-type allele is cut by the Cas9 complex produced by the gRNA encoded by the left construct and the Cas9 encoded by the right construct, causing the right construct to home (copy to the opposite chromosome) using the cell's natural homology-directed repair mechanism. If the construct is not causally related to gene disruption (such as Figure 2 d), then the modification that results in gene disruption should be co-homed with the construct. In another configuration, the left construct can encode its own Cas9 or other RNA-guided nuclease. Note that a second construct containing an enhancer gRNA can be co-homed with Figure 2 Used together with any of the molecular configurations shown in , will have a similar effect of increasing the amount of release.

[0225] Alternatively, the second construct can encode two gRNAs, one that allows homing of the first construct and another that allows homing of the second construct in the presence of the first construct, creating a dual driver that can predictably spread to other populations and whose spread can be controlled by exploiting pre-existing sequence differences between target and non-target populations ( Figure 4 ).

[0226] refer to Figure 4 a, Shows an example of a dual-actuated molecular configuration, where Figure 2 a The genetic construct shown on the left (here shown as the α construct) is paired with a second construct (β) encoding two gRNAs, one targeting the insertion site of the α construct (green) and one targeting the wild-type allele of its own insertion site (grey). Figure 2 As shown in a, the orange arrow shows that the editor acts on the target sequence downstream of the editor insertion site on the two chromosomes to produce a dominant mutation, which causes infertility or lethality in a wild-type background. Alternatively, the target site on the chromosome carrying the construct can be independently modified to produce a dominant negative mutation (due to the early termination codon introduced by the construct and not expressed), and then when the chromosome is cut by the editor, the mutation is homed to the chromosome that does not carry the construct. In addition, the green and gray arrows indicate that in organisms that are heterozygous for α constructs and heterozygous for β constructs, the sequence at the insertion site of each construct on the wild-type allele is cut by the Cas9 complex produced by the gRNA encoded by the β construct and the Cas9 encoded by the α construct, resulting in the homing of the two constructs. Each construct's respective homing can also occur in individuals that are homozygous for another construct. If the beta construct is inserted into a differentiated locus (diff), where some chromosomes have a sequence recognized by the gray gRNA and some do not (and are therefore resistant to cleavage), and the frequencies of the two types of chromosomes are different in different populations, localization of the construct and population suppression can be achieved as long as the resistance frequency is low enough in the target population, allowing both constructs to spread, but high enough in the non-target population to prevent the beta construct from spreading. In another configuration, the beta construct can encode its own Cas9 or other RNA-guided nuclease, allowing the beta construct to home in the absence of the alpha construct. The beta construct can be inserted into the diff site with the alpha construct. Figure 2 Any of the molecular configurations shown in can be paired to produce dual actuation with similar effects.

[0227] Alternative methods of enhancing the first construct may require it to include additional elements; for example, the first construct may carry a rescue construct to mask the effects of the mutation produced by the second construct. Other constructs may also be included to increase the frequency of the second construct.

[0228] The inventors also proposed an alternative molecular design to achieve Figure 2 Feature a) of , wherein the construct is configured to integrate at a location outside the haplosufficient gene (near or more distantly linked thereto) and is designed to induce a mutation in the haplosufficient gene that results in recessive sterility or lethality, for example, by including in the construct a second gRNA that targets the haplosufficient gene (see Figure 11 ).

[0229] like Figure 11As shown, localized population suppression (or non-localized partial or complete suppression) can be achieved using the gene constructs of the present invention, wherein a second gRNA in the construct is used to induce a recessive effect of the construct. Figure 11 a shows the use of a single HS ETDN The molecular mechanism of genes (similar to Figure 2 Design on the left in a). Here, the construct (shown as Cas9 and two gRNAs) encodes a genome editor that: i) targets HS ETDN The wild-type allele of the gene results in dominant lethality or sterility in both sexes; ii) targeting the allele linked to the construct and located upstream of the dominant mutation to produce an early stop codon, resulting in recessive sterility or lethality in both sexes. The presence of the early stop codon prevents the expression of the downstream dominant negative mutation. Therefore, individuals that inherit a copy of the construct and the stop codon associated with it, and a wild-type allele have a fitness close to normal (although if they are homozygous for the construct and the stop codon associated with it, they are not viable or sterile). By recoding the allele linked to the construct so that it is still functional but can be targeted by the genome editor, while the WT allele is unaffected, specificity for the allele linked to the construct can be achieved.

[0230] like Figure 11 As shown in Figures 11b and 11c, the gene constructs of the present invention can also be configured to induce recessive sterility or lethal mutations in a second gene that is independent of the gene targeted to induce dominant sterility or lethal mutations. In this case, the construct can be linked to the HI gene or any gene that can produce dominant lethal mutations or sterility mutations in both sexes or only in females (similar to Figure 2 The construct in b, such as Figure 11 b) or unlinked (similar to Figure 2 c constructs, such as Figure 11 c), and in both cases the construct can be linked or unlinked to the HS gene targeted to induce a recessive lethal or sterility mutation.

[0231] Example 2 - Complete Inhibition

[0232] 2.1 Single locus screening

[0233] Single-locus screening revealed 12 strategies that achieved complete eradication after a single release ( Figure 5 ). In this case, the construct is recessive sterile or lethal and the edit is dominant, and the allele carrying the construct is protected from the dominant edit due to the presence of a premature stop codon or the target site has been recoded or removed, but the sex-specificity is different from the localized control. Figure 5 Also shown are time series simulations of the relative female population size following a single release of a construct designed to use a single locus (released in heterozygotes at a ratio of 50% of the initial male population size).

[0234] If construct and gene disruption affect only one sex, then editing should affect the sex identical with construct (fFF, mMM), or editing should be double sex (fBB, mBB), or editing should be double sex and provide partial protection in trans case, so that the sex affected in construct / editing heterozygote is the same as the sex in construct / construct homozygote (fBF, mBM). In the last case, even when editing occurs only in one sex, as long as the sex is the sex that bears recessive fitness effect due to construct insertion, complete inhibition can be achieved. Alternatively, if construct and gene disruption affect both sexes, then editing should also be double sex, and in trans case, sex-specific protection should be present (i.e., construct / editing heterozygote is sterile / lethal only in one sex) (bBF, bBM).

[0235] When individual heterozygotes for a construct or edit incur some cost (which would otherwise be completely recessive) and editing is suboptimal ), only three designs were able to suppress more than 99%. These designs include strategies 3, 5, and 9 (fBFB, fBBB, and fFmFB). If editing is only possible in one sex, strategies 7 and 8 are suitable.

[0236] 2.2 Molecular configuration /

[0237] These requirements can be met by using appropriate combinations of genes and exons required in only one sex (due to sex-specific splicing), incorporating sequences into constructs that ensure they are spliced ​​out in one sex but not the other, and modifying control regions on genes to either express them in only one sex or to increase expression to provide partial (sex-specific) protection ( Figure 6 ).

[0238] refer to Figure 6 A and B show examples of single-locus molecular configurations that can be used for complete self-sustaining repression. In each configuration, if the terms "female" and "male" are swapped, the strategy still works, albeit with different kinetics. Figure 6 aThe left side shows Figure 5 Here, a construct (shown here as Cas9 and gRNA) is inserted to disrupt the female-specific haploid gene (HS ETDN, pink). Arrows indicate that in organisms heterozygous for the genomic construct and the wild-type allele of the female-specific HS gene, the editor acts on target sequences located downstream of the editor insertion site on both chromosomes to generate dominant negative mutations that cause lethality or sterility in a wild-type genetic background. Alternatively, as Figure 6 a As shown on the right, the construct can be inserted into the female-specific exon (HS) of the haplosufficient gene (HS, grey) for both sexes. ETDN , pink), so that gene function is mainly destroyed in females, and the genome editor is configured to produce a dominant negative mutation at the target site downstream of the destruction in the same sex-specific exon or in another sex-specific intron of the same gene. The pink and blue thin lines show the sex-specific splicing pattern of the gene; for simplicity, they are only shown on the upper chromosome, but also apply to the following (wild type) alleles. In both cases (left and right), when the editor is located downstream of the destruction, due to the presence of premature termination codons, the mutation on the chromosome carrying the construct is not expressed, thereby protecting the construct. However, the target site can also be located upstream of the destruction, in which case the target site on the chromosome containing the construct must be modified or removed so that it is not recognized by the editor.

[0239] Figure 6 b shows an example of the molecular design of strategy 3 fBB (strategy 4 [mBB] if sex is reversed), where the disruption is sex-specific and the dominant negative mutation generated by the editor affects both sexes. Left: The construct is inserted to disrupt the female-specific exon (HS, pink) of a haplosufficient gene and targets the second exon (HS) of the same gene that is expressed in both sexes. ETDN , gray). Right: Alternatively, the construct is inserted into a haploid-sufficient gene (HS ETDN , gray), but with splice control sequences (not shown) that cause the construct to be spliced ​​out in males, so that disruption primarily affects females. In both cases, the target site on the chromosome carrying the construct has been recoded so that it is not recognized by the editor (rHS ETDN ; no orange arrow).

[0240] Strategies 3 and 4 can also be implemented using two independent loci ( Figure 6c), where the genome editor is configured to create a dominant mutation in a gene closely linked to the gene disrupted by the construct (indicated by the arrow). A construct can be inserted to disrupt a female-specific haplosufficient gene (left; HS, pink) or a haplosufficient gene required for both sexes (HS, gray), but the construct contains sequences that ensure its excision in males (right). Genome editors can be designed to create a knockout mutation in a haploinsufficient (HI) gene or to create a dominant negative mutation in another gene (Gene ETDN , gray). In both cases, the target site on the chromosome containing the construct can be recoded so that it is protected from the editor (rHI / rGene ETDN ; no orange arrow).

[0241] Figure 6 d shows Figure 5 Example of configuration of strategy 11 in Figure 2 (strategy 12 if the sexes are opposite). Here, the construct is inserted and disrupts a haploid sufficient gene (HS, grey) required for both males and females, and the editor creates a dominant mutation in a gene that is required for both sexes and is tightly linked to the disrupted gene. Left: The editor targets a gene to create a dominant negative mutation. The chromosome containing the construct is modified so that the gene containing the target site is not affected by the editor (rGene). ETDN ) recognition but still has function, and expression is increased in males, so that female heterozygotes of only editing and construct are lethal or infertile (blue arrows indicate male-specific enhancement). Alternatively, the construct can also encode a module that is expressed only in males and play a role in invalidating the dominant negative mutation effect, rather than increasing the expression of the modified gene in males. This can be an RNA editor (RNAe), which will restore the RNA transcribed from the editing gene to wild type, or it can be an RNA interference (RNAi) module that can degrade it. Right: Alternatively, the editor is configured to produce knockout mutations in tightly linked haploinsufficient genes (HI, gray) required for both sexes. The chromosome containing the construct is modified so that the gene containing the target site is not recognized by the editor (rHI) but still has function, and comprises a second copy of the recoded target gene, the second copy also comprising a sequence that ensures that it is expressed only in males, thereby ensuring that males heterozygous for the construct and editing have normal fitness, while females are infertile or lethal. As shown here, male-specific expression can be achieved by using a suitable promoter (blue arrow) or inserting an intron that will be spliced ​​out only in males and introducing a premature stop codon in females. Alternatively, instead of incorporating a second copy of a modified gene that is expressed only in males, the construct can contain a module that negates the effects of the haploinsufficient knockout (e.g., an RNA editor).

[0242] 2.3 Double locus screening

[0243] These requirements can also be met by using two closely linked loci or two distantly linked or unlinked loci. Designs 1 to 4 can also be implemented using a two-locus model, where editing is performed at unlinked loci and the construct fully rescues the dominant fitness effect of the editing (kinetics similar to those of Figure 7 ). If editing is only possible in one sex, strategies 7 and 8 are still useful and more efficient than when using a single locus, as are strategies 13 and 14.

[0244] 2.4 Improve efficiency

[0245] If the construct also catalyzes its own homing reaction, the rate at which the construct spreads through the population and inhibits the population may be increased. Even a lower homing rate can still speed up the rate (or reduce the release rate required to achieve inhibition in a specific time frame).

[0246] Example 3 - Partial Inhibition

[0247] Another use of the present invention can be to provide non-localized (self-diffusion) partial control. This may be useful if the ultimate goal of the control plan is partial control, or if the ultimate goal is full control but it is desired to be achieved in a step-by-step manner as part of a risk mitigation strategy. The inventors are not aware of any other proposed strategies that provide self-diffusion partial control when the fitness effect and editing rate are ideal (i.e., 0 or 1).

[0248] 3.1 Single locus screening

[0249] Single-locus screening identified 26 strategies with equilibrium levels of suppression ranging from 50% to 99% ( Figure 8 In this case, the construct can be associated with a recessive sterility or lethal effect in both sexes or in only one sex. If the effect is bisexual ( Figure 8 To achieve strategies 1 to 4 of these approaches, the construct must provide dominant protection against editing, regardless of whether the editing is in cis or trans. This can be achieved by introducing into the construct modules that nullify the editing effect at the RNA level, such as RNA editors (which restore the RNA expressed from the edited gene to the wild-type sequence) or RNAi modules (which degrade the editing RNA).

[0250] In the absence of the construct, editing should result in dominant lethality or sterility in both sexes (strategy 1 [bB-]), females only (strategy 3 [bF-]), male-recessive lethal / sterile females (strategy 4 [bFm-]), or female-recessive lethal / sterile males (strategy 2 [bfM-]). Figure 8Shown is the time course of relative female population sizes following a single release of these constructs in male heterozygotes at a proportion of 50% of the initial male population, where the design that achieved suppression most rapidly involved editing in both sexes (Strategy 1 [bB-]).

[0251] Figure 10 a shows Figure 8 Here, a construct (shown here as Cas9 and gRNA) is inserted into a haploid-sufficient gene (HS ETDN , gray). Arrows indicate that in organisms heterozygous for the wild-type alleles of the genome editor and the HS gene, the editor acts on the wild-type target sequence on the chromosome that does not carry the construct, resulting in a dominant negative mutation that is lethal or sterile in both males and females (orange arrow); the target site on the chromosome carrying the construct has been recoded so that it is still functional but not recognized by the editor (rHS ETDN ; No orange arrows); The construct also encodes an RNA editor (RNAe) or RNA interference (RNAi) module, which plays a role in invalidating the editor effect and providing dominant rescue. In a wild-type genetic background, individuals carrying more than one editing target site are lethal or sterile. In individuals carrying at least one copy of the editing target site and a construct, the editing RNA (gray line, editing is shown in orange) transcribed from the gene carrying the editing allele is degraded (by the effect of the RNAi module encoded by the construct) or restored to wild type (gray line, by the effect of the RNA editor encoded by the construct) before RNA translation, thereby protecting the individual from the influence of dominant negative mutations. RNA transcribed from the chromosome carrying a copy of the recoded target gene (gray line, recoded sequence is shown in gray) is resistant to RNAe or RNAi module activity. Please note that here (and in all other constructs containing RNAe / i modules as described below), if the construct encodes an RNA editor, there is no need to recode the target site on the construct chromosome, because the RNA transcribed therefrom will also be restored to wild type. Figure 8 Strategies 2 to 4 can be used as follows Figure 10 Similar molecular configurations as shown in a can be constructed, for example, with the target site located in a sex-specific intron. Strategies 1 to 4 can also be used with other dual-locus molecular designs, for example, with the genome editor target site located in a separate gene, near the disrupted gene (meiotic recombination rate <1%), or distant from the disrupted gene (meiotic recombination rate >1%).

[0252] Efficiency can be improved if the construct is associated with a female-specific recessive lethal or sterile gene, provides dominant protection against the edits it produces (e.g., by RNA editing or RNA interference), regardless of its position relative to the construct integration site, and the genome editor is designed to produce dual-sex dominant edits (strategy 5 [fB-]) or female-specific edits (strategy 9 [fF-]). Although the same equilibrium state inhibition level can be achieved, it will take longer to achieve if the edits occur in only one sex (strategies 6 and 7), are dual-sex recessive (strategy 8), or result in some male cost (strategies 10-13).

[0253] Figure 10 b and c show Figure 8 Examples of molecular design of strategies 9 and 5 in

[15] , which only involve a single gene. Figure 10 b (left side) shows the realization of Figure 8 One approach in strategy 9 is to integrate the gene construct into the female-specific haplosufficient gene (HS ETDN , pink) and disrupt it. Alternatively, the gene construct is integrated into and disrupted in the female-specific exon of a haploid-sufficient gene required for both sexes, and the genome editor generates sex-specific ( Figure 10 b[right]; HS ETDN , pink; strategy 9) or dual gender ( Figure 10 c[left]; HS ETDN , gray; dominant negative mutations or gain-of-function mutations of strategy 5). Figure 10 c (right), another way to implement strategy 5 is to integrate the gene construct into the double sex haploid sufficient gene (HS ETDN , gray) and disrupt it, and make the gene construct contain sequences that ensure it is spliced ​​out in a sex-specific manner. Similarly, the pink and blue thin lines show the sex-specific splicing patterns of the gene; for simplicity, they are only shown on the upper chromosome, but also apply to the lower (wild-type) allele. In each case, the construct must function to negate the editor effect and provide dominant rescue (RNAe / i), and the target site on the chromosome carrying the construct should be recoded so that it is still functional but not recognized by the editor (rHS ETDN ). Figure 8 Strategies 6 to 8 and 10 to 13 can be used as follows Figure 10 Similar molecular configurations as shown in b and c can be constructed where, for example, editing occurs only in one sex and / or the sex specificity of the edited alleles differs from that in strategies 5 or 9.

[0254] Alternatively, strategies 5 to 13 can be implemented using two closely linked genes. Figure 10e and f show Figure 8 Example of a dual-locus molecular design for strategies 9 and 5, where the genome editor targets the wild-type allele of another gene near the disrupted gene. Figure 8 One approach to strategies 5 and 9 is to integrate the gene construct into and disrupt the female-specific haplosufficient gene (HS, pink) ( Figure 10 e[left] and Figure 10 f [left]), or by integrating a gene construct into a sex-specific haploid-sufficient gene for disruption and including sequences that ensure its excision in a sex-specific manner ( Figure 10 e[right] and Figure 10 f [right]). Similarly, in each case, the construct can also function to nullify the editor effect and provide dominant rescue (RNAe / i), and the target site on the chromosome carrying the construct has been recoded so that it remains functional but is not recognized by the editor (rHI / rHS ETDN ). Figure 8 Strategies 6 to 8 and 10 to 13 can be used as follows Figure 10 Similar molecular configurations as shown in b and c can be constructed, for example, where editing occurs only in one sex and / or the sex specificity of the edited alleles differs from that in strategies 5 or 9.

[0255] Alternatively, if the construct is sex-specific and the genome editor produces a dominant edit affecting the opposite sex ( Figure 8 , strategy 14 [fMM], strategy 18 [ffMM], strategy 22 [mFF], and strategy 23 [mFmF]), partial inhibition can be achieved without the need for the construct to provide explicit protection against editing (i.e., contain an RNA editing or interference module). Figure 10 d shows an example of a molecular configuration for strategy 22 (fMM), using a single gene with male and female sex-specific exons, such as doublesex. Here, a construct is inserted that disrupts the male-specific exon (HS, blue), causing the construct to be spliced ​​out in females, so that only male homozygotes are lethal or sterile. The genome editor is configured to insert a gene at the female-specific exon (HS ETDN , pink) produces a dominant mutation in which males are sterile or lethal in a wild-type background. The construct must also protect the target site located on the same chromosome, for example by modifying or removing the target site so that it can no longer be edited by the editor (rHS ETDN ) identification. Strategy 14 can be implemented in a similar manner, where Figure 10 The genders are swapped in the example shown in d. Figure 10g shows an example of a two-locus molecular design for strategy 14, in which the genome editor acts on the wild-type allele of another gene that is closely linked to the disrupted gene. Here, the gene construct is integrated into and disrupts a female-specific haplosufficient gene (HS, pink; left) or a bisexual haplosufficient gene (HS, gray; right), and the gene construct contains sequences that ensure its excision in a sex-specific manner. The editor can generate a dominant knockout mutation in a male-specific haploinsufficient gene (HI, blue) or a dominant negative mutation in any possible gene (HS ETDN , blue). Similarly, the construct can also protect the target site located on the same chromosome. If the editor produces a knockout of the HI gene, the gene can be a recoded version thereof, which still has function but is not recognized by the editor (rHI), and if the editor is dominant negative, it can only involve the removal of the gene. Strategy 18, 22 and 23 can be implemented using a molecular design similar to strategy 14, wherein the sex specificity and / or splicing of the gene are different from strategy 14.

[0256] For each design, higher levels of suppression could be achieved if a second construct of the same design located elsewhere in the genome was released into the same population after the first release achieved suppression. Figure 8 This is demonstrated by the six most efficient designs in c and d.

[0257] 3.2 Double locus screening

[0258] In two-locus screens simulated with idealized and non-idealized parameters, eight strategies were found. In both single-locus and two-locus screens, two strategies (strategy 22 [mFFB] and strategy 14 [fMMB]) were common. Figure 10 h shows Figure 8 An example of a dual-locus molecular design for strategy 14, in which the genome editor targets the wild-type allele of another gene distant from the disrupted gene. Here, the gene construct is integrated into the sex-specific haplosufficient gene ( Figure 10 h [left]) or a bisexual haplosufficient gene and disrupting it, and including sequences in the gene construct that ensure its excision in a sex-specific manner ( Figure 10 h). In each case, the construct can also encode a recoded version of the target gene, making it resistant to editing, while carrying a single copy of the dominant edit (rHI / rGene ETDNFor strategies 14 and 22, if the genome editor acts on the wild-type allele of another unlinked gene, the strategy is still effective if editing occurs only in one sex (indicated by a blue arrow and the initials of the sex in which expression occurs), although with slightly different kinetics ( Figure 9 , strategies 27, 29, 31, and 32).

[0259] Finally, dual-locus screens revealed two additional strategies (28 and 30) in which constructs are sex-specific and genome editors produce edits that affect the sex opposite to the sex affected by the disruption. This can be achieved by expressing Cas9 from a promoter that is active only in a single sex. Figure 10 i shows an example of a dual locus molecule design for strategy 28. Similarly, the construct can encode a recoded version of the target gene for use in a gene carrying a single copy of the dominant edit (rHI / rGene ETDN ) individuals.

[0260] in conclusion

[0261] The inventors have discovered a novel genetic construct that can disrupt haplosufficiency genes required for survival or reproduction in male and / or female organisms. Furthermore, this genetic construct encodes a genome editor that produces a dominant lethal or sterile phenotype in males and / or females, rendering individuals carrying the mutation infertile. Advantageously, this genetic construct can be inserted into an organism and released into a population to effectively suppress the population over a period of time.

[0262] In summary, the gene constructs of the present invention differ from the gene control elements previously used to control pest populations in that:

[0263] - gRNA and Cas9 are designed to induce a dominant mutation at a locus outside the construct insertion site, which may or may not be located in a haplosufficient gene.

[0264] - The construct is designed to provide protection against a single copy of the dominant mutation it generates.

[0265] - The gRNA and Cas9 in the claimed gene construct do not result in homing of the construct, and population suppression is expected to be achieved without homing.

[0266] The inventors believe they are the first to design a gene construct that: (i) causes recessive sterility or lethality; (ii) produces a dominant mutation; and (iii) provides protection against dominant mutations.

[0267] Advantageously, the inventors have discovered that, depending on the precise configuration, the genetic constructs can produce three different types of potentially useful effects when released into a target population: localized inhibition, non-localized partial inhibition, or non-localized complete inhibition.

Claims

1. A genetic construct comprising a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence is configured to disrupt a haplosufficient gene required for survival or reproduction of a male and / or female organism, or is configured to integrate into or near a disrupted allele of the haplosufficient gene, and the second nucleotide sequence encodes a genome editor that generates a dominant lethal mutation or a sterility mutation in a target gene expressed in a male and / or female organism, such that: a homozygote of the genetic construct is lethal or sterile, and / or a heterozygote of the dominant lethal mutation or sterility mutation is lethal or sterile.

2. The gene construct according to claim 1, wherein The genetic construct is configured to disrupt the haplosufficient gene by integrating the genetic construct into the haplosufficient gene at an integration site or a disruption site, and optionally, the genetic construct introduces a premature stop codon into the haplosufficient gene.

3. The gene construct according to claim 2, wherein The gene construct is integrated into the region between the 5' promoter and the 3' end of the haplosufficient gene, or the gene construct is integrated between exons or introns of the haplosufficient gene.

4. The gene construct according to claim 1, wherein The genetic construct is configured to integrate into the disrupted allele of the haplosufficient gene, or is configured to integrate at a position outside but near the disrupted allele of the haplosufficient gene, preferably is configured to integrate within 10, 8, 6, 4, 2 or 1 centimorgan of the haplosufficient gene, optionally, the haplosufficient gene is disrupted by the introduction of a knockout mutation, optionally by the introduction of a premature stop codon, or by a complete or partial deletion of the haplosufficient gene.

5. A genetic construct according to any one of the preceding claims, wherein The genetic construct is configured to disrupt a haplosufficient gene required for survival or reproduction of male and female organisms, or is configured to integrate into or near the disrupted allele of the haplosufficient gene such that males and females homozygous for the genetic construct are lethal or sterile.

6. A genetic construct according to any one of the preceding claims, wherein The genome editor generates a dominant lethal mutation or a sterility mutation in a target gene expressed in female organisms, or in a target gene expressed in both male and female organisms.

7. A genetic construct according to any one of the preceding claims, wherein The target gene is the wild-type allele of the disrupted haplosufficient gene.

8. A genetic construct according to any one of the preceding claims, wherein The genome editor generates a dominant negative or dominant gain-of-function mutation in the wild-type allele of the disrupted haploid-sufficient gene.

9. The gene construct according to claim 7 or 8, wherein The genome editor targets a site in the haplosufficient gene that is downstream of the integration site, or the genome editor targets a site in the haplosufficient gene that is upstream of the integration site.

10. A genetic construct according to any one of the preceding claims, wherein The genome editor generates a dominant negative or dominant gain-of-function mutation in a female-specific exon of a haplosufficient gene required for male and female organisms, optionally a homolog of the Drosophila genes doublesex or fruitless.

11. The gene construct according to any one of claims 1 to 6, wherein The target gene is a gene located near the disrupted haplosufficient gene.

12. The gene construct according to claim 11, wherein The genome editor generates a dominant negative or dominant gain-of-function mutation in a wild-type allele of a target gene located near a disrupted haplosufficient gene, or the genome editor generates a knockout mutation in a wild-type allele of a haploinsufficient target gene located near a disrupted haplosufficient gene.

13. The gene construct according to claim 11 or 12, wherein The meiotic recombination rate of the target gene and the disrupted haplosufficient gene is less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

14. A genetic construct according to any one of the preceding claims, wherein The target site on the chromosome containing the construct is modified or removed so that it is not recognized by the genome editor.

15. The gene construct according to any one of claims 1 to 6, wherein The target gene is a gene located distal to the disrupted haplosufficient gene.

16. The gene construct according to claim 15, wherein The genome editor generates a dominant negative or dominant gain-of-function mutation in a wild-type allele of a target gene that is distal to the disrupted haplosufficient gene, or the genome editor generates a knockout mutation in a wild-type allele of a haploinsufficient target gene that is distal to the disrupted haplosufficient gene.

17. The gene construct according to claim 15 or 16, wherein The meiotic recombination rate of the target gene and the disrupted haplosufficient gene is greater than 0.5%, greater than 1%, greater than 2%, greater than 3%, greater than 4% or greater than 5%.

18. A genetic construct according to any one of the preceding claims, wherein The gene construct also comprises a nucleotide sequence encoding a rescue copy of the target gene.

19. The gene construct according to any one of claims 1 to 4, wherein The genetic construct is configured to disrupt a haplosufficient gene required for survival or reproduction of a male or female organism, or is configured to integrate into or near a disrupted allele of a haplosufficient gene such that males or females homozygous for the genetic construct are lethal or sterile.

20. The gene construct according to claim 19, wherein The genetic construct comprises a nucleotide sequence encoding a genome editor that produces a dominant lethal mutation or a sterility mutation in a target gene expressed in male and / or female organisms.

21. The gene construct according to claim 19 or 20, wherein The target gene is the wild-type allele of the disrupted haplosufficient gene.

22. The gene construct according to any one of claims 19 to 21, wherein The genome editor generates a dominant negative or dominant gain-of-function mutation in the wild-type allele of the disrupted haplosufficient gene, or the genome editor generates a dominant negative or dominant gain-of-function mutation in the wild-type allele of a target gene located near the disrupted haplosufficient gene, or the genome editor generates a knockout mutation in the wild-type allele of a haploinsufficient target gene located near the disrupted haplosufficient gene.

23. A gene construct according to any one of claims 19 to 22, wherein The genome editor generates a dominant negative mutation or a dominant gain-of-function mutation in a female-specific haplosufficient gene, optionally selected from homologs of the Drosophila genes ovo, dorsal, torso, easter, and Toll.

24. The gene construct according to any one of claims 19 to 22, wherein The genome editor generates a dominant negative mutation or a dominant gain-of-function mutation in a male-specific haplosufficient gene, optionally selected from homologs of the Drosophila genes betaTub85D and whirligig.

25. The gene construct according to any one of claims 19 to 22, wherein The disrupted haplosufficient gene is disrupted at a female-specific exon of the haplosufficient gene. Alternatively, the disrupted haplosufficient gene is disrupted at a female-specific exon of a doublesex or fruitless gene, or at a female-specific exon of a homolog of a doublesex or fruitless gene.

26. The gene construct according to any one of claims 19 to 22, wherein The disrupted haplosufficient gene is disrupted at a male-specific exon of the haplosufficient gene, alternatively, the disrupted haplosufficient gene is disrupted at a male-specific exon of a doublesex or transformer gene, or at a male-specific exon of a homolog of a doublesex or transformer gene.

27. The gene construct according to any one of claims 19 to 26, wherein The genetic construct comprises a nucleotide sequence that ensures that the construct will be spliced ​​out of the RNA of the male organism. Alternatively, the genetic construct comprises a nucleotide sequence encoding a splicing control sequence of a homologue of the transformer, doublesex or fruitless gene, which splicing control sequence ensures that the construct will be spliced ​​out of the RNA of the male organism.

28. The gene construct according to any one of claims 19 to 26, wherein The genetic construct comprises a nucleotide sequence that ensures that the construct will be spliced ​​out from the RNA of the female organism. Alternatively, the genetic construct comprises a nucleotide sequence that encodes a splicing control sequence of a homolog of the Drosophila transformer gene, which splicing control sequence ensures that the construct will be spliced ​​out from the RNA of the female organism.

29. The gene construct according to any one of claims 19 to 28, wherein The target site on the chromosome containing the construct is modified or removed so that it is not recognized by the genome editor.

30. The gene construct according to claim 29, wherein Because the target site has been recoded, the chromosome containing the gene construct is protected from the genome editor and the expression of the recoded target gene is enhanced in a sex-specific manner, or the chromosome containing the gene construct contains a second copy of the recoded target gene with control sequences that ensure its expression only in males or females, so that only male or female heterozygotes containing the construct and the mutation are affected.

31. The gene construct according to any one of claims 1 to 4, wherein The gene construct comprises a nucleotide sequence encoding an RNA editor or an RNA interference module. Preferably, the RNA editor or RNA interference module restores the RNA expressed from the mutant gene to the wild-type sequence by RNA editing, or removes the RNA transcribed from the edited gene by RNA interference (RNAi).

32. The gene construct according to claim 31, wherein The RNA editor or the RNAi module is expressed in only one sex.

33. The gene construct according to any one of claims 1 to 4, wherein The genetic construct is configured to disrupt a haplosufficient gene essential for fertility or survival of a male or female organism, or is configured to integrate into or near the disrupted allele of the haplosufficient gene, and the genome editor generates a dominant negative or dominant gain-of-function mutation in the wild-type allele of the same locus, wherein the dominant negative or dominant gain-of-function mutation affects the sex opposite to the sex in which the gene disruption was performed.

34. The gene construct according to claim 33, wherein The disrupted haplosufficient gene is disrupted at a male-specific exon, resulting in recessive male-specific lethality or sterility, and the genome editor generates a dominant sterility mutation in a female-specific exon. Optionally, the target gene for disruption is a homolog of doublesex.

35. The gene construct according to claim 33, wherein The disrupted haplosufficient gene is disrupted at a female-specific exon, resulting in recessive female-specific lethality or sterility, and the genome editor generates a dominant sterility mutation in a male-specific exon. Optionally, the disrupted target gene is a homolog of doublesex.

36. The gene construct according to any one of claims 1 to 4, wherein The genetic construct is configured to disrupt a haplosufficient gene essential for fertility or survival of a male or female organism, or is configured to integrate into or near the disrupted allele of the haplosufficient gene, and the genome editor targets the wild-type allele of the target gene located near the disrupted haplosufficient gene, generating a dominant negative mutation, a gain-of-function mutation, or a knockout of the haploinsufficient gene.

37. The gene construct according to any one of claims 1 to 4, wherein The genetic construct is configured to disrupt a haplosufficient gene essential for fertility or survival of a male or female organism, or is configured to integrate into or near the disrupted allele of the haplosufficient gene, and the genome editor targets the wild-type allele of a gene located near the disrupted haplosufficient gene, resulting in a dominant negative mutation, gain-of-function mutation, or knockout of a haploinsufficient gene that affects the sex opposite to the sex affected by the gene disruption.

38. The gene construct according to any one of claims 31 to 37, wherein The target site on the chromosome containing the construct is modified or removed so that it is not recognized by the genome editor, or the gene construct contains a nucleotide sequence that ensures it will be spliced ​​out in a sex-specific manner.

39. The gene construct according to any one of claims 1 to 4, wherein The genetic construct is configured to disrupt a haplosufficient gene essential for fertility or survival of a male or female organism, or is configured to integrate into or near the disrupted allele of the haplosufficient gene, and the genome editor targets the wild-type allele of a gene located distal to the disrupted haplosufficient gene, resulting in a dominant negative mutation, gain-of-function mutation, or knockout of a haploinsufficient gene that affects the sex opposite to the sex affected by the gene disruption.

40. A genetic construct comprising a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence is configured to integrate into a position outside a haplosufficient gene required for survival or reproduction of a male and / or female organism, and the second nucleotide sequence encodes: (i) a first genome editor, which generates a dominant lethal mutation or a sterility mutation in a target gene expressed in a male and / or female organism, such that heterozygotes for the dominant lethal mutation or the sterility mutation are lethal or sterile; and (ii) a second genome editor, which generates a recessive lethal mutation or the sterility mutation in the haplosufficient gene, such that homozygotes for the recessive lethal mutation or the sterility mutation are lethal or sterile.

41. The gene construct according to claim 40, wherein The second genome editor generates a premature stop codon in the haplosufficient gene.

42. The gene construct according to claim 40 or 41, wherein The first genome editor generates a dominant lethal mutation or a sterility mutation in a haploinsufficient gene or any gene that may generate a dominant lethal mutation or a sterility mutation in both males and females or only in females.

43. The gene construct according to any one of claims 40 to 42, wherein The gene construct also comprises a nucleotide sequence encoding a rescue copy of the target gene.

44. A genetic construct according to any one of the preceding claims, wherein The organism is selected from the group consisting of disease vectors, agricultural pests, or harmful invasive species, optionally including arthropods, other invertebrates, mammals, other vertebrates, and weeds.

45. The gene construct according to claim 44, wherein The arthropod is an insect, arachnid, myriapod or crustacean. Optionally, the insect is a mosquito, a fruit fly of the Tephritidae family, a sand fly, a dipteran insect, a lepidopteran insect, a coleopteran insect or a mealybug.

46. ​​The gene construct according to claim 44, wherein The organism is a gastropod, a bivalve, a fish, an amphibian, a mammal or a plant, preferably a weed.

47. A genetic construct according to any one of the preceding claims, wherein The target gene is the wild-type allele of a disrupted haplosufficient gene, or the target gene is any other gene that can produce a dominant negative or dominant gain-of-function mutation.

48. A genetic construct according to any one of the preceding claims, wherein The target gene encodes a protein that functions as a multimer, or the target gene encodes a transcription factor or a membrane-bound protein, preferably a homodimeric membrane receptor.

49. A genetic construct according to any one of the preceding claims, wherein The target gene is a haploinsufficient gene, optionally, the haploinsufficient gene is a haplolethal gene.

50. A genetic construct according to any one of the preceding claims, wherein The genome editor is selected from the following: a transcription activator-like effector nuclease (TALEN) genome editor, a zinc finger nuclease (ZFN) genome editor, and a CRISPR-based genome editor. Preferably, the genome editor is a CRISPR-based genome editor, most preferably a CRISPR-Cpf1-based or CRISPR-Cas9-based genome editor.

51. A genetic construct according to any one of the preceding claims, wherein The genome editor comprises a first nucleotide sequence capable of hybridizing with a target gene. Preferably, the first nucleotide sequence capable of hybridizing with a target gene is a guide RNA (gRNA).

52. The gene construct according to claim 51, wherein The genome editor further comprises a second nucleotide sequence encoding a CRISPR nuclease, preferably Cpf1 or Cas9 nuclease, most preferably Cas9 nuclease, or a derivative thereof, to allow DNA nicking, base editing, prime editing or other types of editing.

53. The gene construct according to claim 52, wherein The genome editor further comprises at least one promoter sequence that drives expression of the first nucleotide sequence and the second nucleotide sequence, or the genome editor comprises a first promoter sequence operably linked to the first nucleotide sequence and a second promoter sequence operably linked to the second nucleotide sequence.

54. A genetic construct according to any one of the preceding claims, wherein The genome editor further comprises a nucleotide sequence comprising a control sequence that ensures the genome editor is active in the male and / or female germline. Optionally, the genetic construct comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence comprise a first control sequence and a second control sequence.

55. Use of the genetic construct of any one of claims 1-54 for disrupting a haplosufficient gene required for survival or reproduction of a male and / or female organism, or for integration into or near a disrupted allele of said haplosufficient gene, such that homozygotes of said genetic construct are lethal or sterile, and such that a dominant lethal mutation or sterile mutation is produced in a target gene expressed in a male and / or female organism, such that heterozygotes of said dominant lethal mutation or sterile mutation are lethal or sterile, and / or male and / or female organisms comprising said dominant lethal mutation or sterile mutation are infertile.

56. The use of the genetic construct of any one of claims 40 to 43, for integration into a position outside of a haplosufficient gene required for survival or reproduction of a male and / or female organism, and for producing a dominant lethal mutation or a sterility mutation in a target gene expressed in the male and / or female organism, such that a heterozygote of the dominant lethal mutation or the sterility mutation is lethal or sterile, and for producing a recessive lethal mutation or a sterility mutation in a haplosufficient gene expressed in the male and / or female organism, such that a homozygote of the recessive lethal mutation or the sterility mutation is lethal or sterile.

57. A method of producing a genetically modified organism, the method comprising introducing into the organism the genetic construct of any one of claims 1-54.

58. A method for producing a genetically modified organism, the method comprising introducing a genetic construct into the organism, the genetic construct comprising a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence being configured to disrupt a haplosufficient gene required for survival or reproduction of male and / or female organisms, or being configured to integrate into or near a disrupted allele of the haplosufficient gene, the second nucleotide sequence encoding a genome editor, the genome editor generating a dominant lethal mutation or a sterility mutation in a target gene of the male and / or female organism such that: homozygotes for the genetic construct are lethal or sterile, and / or heterozygotes for the dominant lethal mutation or sterility mutation are lethal or sterile.

59. A method for producing a genetically modified organism, the method comprising introducing a genetic construct into the organism, the genetic construct comprising a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence being configured to integrate into a position outside a haplosufficient gene required for survival or reproduction of the male and / or female organism, the second nucleotide sequence encoding: (i) a first genome editor, the first genome editor generating a dominant lethal mutation or sterility mutation in a target gene expressed in the male and / or female organism; and (ii) a second genome editor, the second genome editor generating a recessive lethal mutation or sterility mutation in the haplosufficient gene such that homozygotes for the recessive lethal mutation or sterility mutation are lethal or sterile.

60. The method according to claim 57 or 58, wherein The method further comprises introducing into the organism a second genetic construct comprising a nucleotide sequence configured to increase the frequency of a first genetic construct, wherein the first genetic construct is a genetic construct that disrupts or is associated with disruption of the haplosufficient gene.

61. The method of claim 60, wherein: The second gene construct comprises a nucleotide sequence encoding a guide RNA targeting the integration site of the first gene construct, or the second gene construct comprises a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence encodes a first guide RNA targeting the integration site of the first gene construct and the second nucleotide sequence encodes a second guide RNA targeting the integration site of the second gene construct.

62. A genetically modified organism obtained or obtainable by the method of any one of claims 57 to 61.

63. A genetically modified organism comprising a disrupted haplosufficient gene required for the survival or reproduction of male and / or female organisms, and a nucleotide sequence encoding a genome editor, which generates a dominant lethal mutation or a sterility mutation in the target gene, rendering male and / or female organisms comprising the dominant lethal mutation or the sterility mutation unable to reproduce.

64. A genetically modified organism, comprising a disrupted haplosufficient gene required for survival or reproduction of a male and / or female organism, and a nucleotide sequence encoding a first genome editor that generates a dominant lethal mutation or a sterility mutation in a target gene expressed in the male and / or female organism, such that heterozygotes for the dominant lethal mutation or the sterility mutation are lethal or sterile, and a second genome editor that generates a recessive lethal mutation or a sterility mutation in a haplosufficient gene expressed in the male and / or female organism, such that homozygotes for the recessive lethal mutation or the sterility mutation are lethal or sterile.

65. A method of suppressing a wild-type population of an organism, the method comprising breeding a genetically modified organism comprising a genetic construct comprising a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence is configured to disrupt a haplosufficient gene required for survival or reproduction of male and / or female organisms, or is configured to integrate into or near a disrupted allele of the haplosufficient gene, and the second nucleotide sequence encodes a genome editor that generates a dominant lethal mutation or a sterility mutation in a target gene in the male and / or female organisms such that: homozygotes for the genetic construct are lethal or sterile, and / or heterozygotes for the dominant lethal mutation or sterility mutation are lethal or sterile.

66. A method of suppressing a wild-type population of an organism, the method comprising breeding a genetically modified organism comprising a genetic construct comprising a first nucleotide sequence and a second nucleotide sequence, the first nucleotide sequence being configured to integrate into a position outside of a haplosufficient gene required for survival or reproduction of the male and / or female organism, the second nucleotide sequence encoding: (i) a first genome editor that produces a dominant lethal mutation or sterility mutation in a target gene expressed in the male and / or female organism; and (ii) a second genome editor that produces a recessive lethal mutation or sterility mutation in the haplosufficient gene expressed in the male and / or female organism, such that homozygotes for the recessive lethal mutation or sterility mutation are lethal or sterile.

67. Use of the genetic construct of any one of claims 1 to 54 to suppress a wild-type population of an organism.

68. The method of claim 65 or 66 or the use of claim 67, wherein The inhibition is localized inhibition, non-localized partial inhibition or non-localized complete inhibition.

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