Method for reducing the reproduction of a harmful animal population

Abstract

The present invention relates to a method for reducing the reproduction of a non-human, sexually reproducing, animal population comprising the steps of: (i) providing a genetically modified male carrying a first polynucleotide on the Y chromosome or on an autosome, said first polynucleotide encoding a first population suppression effector, and a second polynucleotide on the X chromosome or on an autosome, encoding a neutralizing element configured to mitigate or remedy the effect of said first polynucleotide; (ii) rearing the genetically modified male of step (i) by allowing it to reproduce with a genetically modified female carrying the said second polynucleotide on a X chromosome or on an autosome, obtaining an animal population wherein the males carry both the first and the second polynucleotide; (iii) removing fertile females from the population obtained in step (ii); (iiia) optionally, rearing the males obtained in step (iii) allowing them to reproduce with a population of non-genetically modified females and isolating the male offspring carrying only the first polynucleotide; (iv) releasing the animals obtained in step (iii) or the male offspring obtained in step (iiia) into the environment where a reduction of the reproduction of the animal is sought, obtaining a reduction of the reproduction of the animal population.

Description

[0001] METHOD FOR REDUCING THE REPRODUCTION OF A HARMFUL ANIMAL POPULATION

[0002] The present invention relates to a method for reducing the reproduction of a non-human, sexually reproducing, diploid animal population.

[0003] There is a pressing need to control the spread and impact of animal pest and vector species that cause increasing biological and socio-economic damage to the environment, agriculture, and human health (Bernaola & Holt, 2021; Dent & Binks, 2020; Edde, 2021; Swei et al., 2020). These species include invertebrate pests attacking important crops, arthropod vectors of human, animal, or plant disease, and invasive animal species that threaten important natural ecosystems. Establishing effective control measures for biological control, such as those targeting arthropod pest or vector populations, is a complex, continuing, and difficult problem, and new technologies need to be developed to reduce the incidence of vector-borne diseases, as well as the destruction of agricultural crops and ecosystem balance by invasive species. Amongst the methods employed for controlling animal pest populations, for example arthropods, are the genetic modification of the crop species that are damaged, inducing the expression of toxins that are harmful to the animals damaging them, the use of broadspectrum pesticides, baited traps and poisons, hunting, or the introduction and release of natural predators. While these methods can be generally effective, animals, and particularly pest and / or invasive arthropods or disease vectors, continuously evolve resistance mechanisms against most of these solutions, resulting in the need for developing new technologies (Barzman et al., 2015; Childers et al., 2021; Onstad & Knolhoff, 2014). Additionally, blanketing crops or eliminating disease vectors with insecticides or poisons is not cheap, is damaging to the environment, and not sustainable in the long term. Furthermore, the introduction of insecticidal traits into all crops under serious threat of destruction by pest and / or invasive arthropods is not feasible, extremely expensive, faces issues of social acceptance, and cannot solve all problems related to arthropod pests (Bawa & Anilakumar, 2013). Furthermore, even though the release of natural predators can constitute an effective method for controlling some animal pest and / or invasive species or disease vectors, the use of this strategy remains limited due to its environmental risks, elevated cost, lack of availability of suitable natural predators, and regulation (Barratt et al., 2018).

[0004] Given the limitations and generality of action of broad-spectrum techniques for the control of pest and / or invasive animals or disease vectors, the targeted modification of pest and / or invasive species or disease vectors is considered a promising approach to use in the field. In the techniques employing this approach, the modified animals are released in the area of interest and suppress or modify populations of their wild counterparts through mating. Various technologies are used to modify the released animals, including radiation, chemical, genetic and microbiota-based interventions. Technologies based on animal modification present multiple advantages, a chief one being that their effects only and specifically affect the pest or disease vector species, therefore leaving the rest of the ecosystem relatively untouched.

[0005] In theory, technologies that are based on animal modification could be effective through the release of either male or female effector animals, herein defined as those individuals modified to impair reproduction of an animal population. To date, control programs based on such strategies have largely entailed male-only releases for two main reasons. First, because females are disproportionately responsible for the harm caused by pest and / or invasive arthropod species or disease vectors. For mosquitoes and several other blood-sucking arthropods, only the females take blood meals and as a consequence, only the females transmit disease. In other pest and / or invasive species or disease vectors, the damage is often caused by egg laying, as is the case for the invasive pest Drosophila suzukii, which lays its eggs inside freshly ripe fruit. Secondly, females are the limiting determinant of the reproductive capacity in an animal population, and thus strategies that aim to block female reproduction tend to be more effective than those targeting male reproduction.

[0006] Sterile insect technique (SIT), based on mass-production and release of sterile males, has historically been used to control, and eradicate, insect pest populations dating back to the mid-1930s (Dyck et al., 2021). This technique has shown to be species- specific and environmentally friendly and has laid the grounds for large-scale operations for the successful control of insect populations (Benedict & Robinson, 2003). Nonetheless, SIT displays several limitations. First, since the released males are sterile, the effects of an SIT intervention last only one insect generation. For this reason, long-lasting SIT suppression efforts rely on the continuous release of such males, which can represent a costly and complex endeavour. Additionally, SIT relies on the release of adult males, which is more costly than releasing insects at earlier life stages (e.g. eggs and larvae). It is also important to underscore that a major operational challenge of SIT is that it requires sex sorting, i.e., the separation of males and females prior to release. While there have been recent advances in this respect, including image-based (i.e., Verily Life Sciences (Bouyer et al., 2022; Crawford et al., 2020), size-based (Oxitec (Carvalho et al., 2014), among others), and fluorescence-based (COPAS (Marois et al., 2012)) sex-sorting, these solutions remain expensive, can be inaccurate, can be difficult to establish in the release areas where they are needed, and can cause damage to the reared males. Though sex separation does not necessarily need to be complete for these strategies to be effective, male-only releases typically aim to release a population in which the females are fewer than 5%. Finally, the treatments that are traditionally used to induce sterility of SIT males, such as ionizing radiation and some chemicals, often further (and heavily) exacerbate the fitness costs incurred by males through the sex-sorting process. Such costs reduce mating competitiveness of released males in the wild and therefore the overall efficacy of the approach, which is often compensated for by increasing the number of released males. In turn, this increases the operational complexity and cost of each release.

[0007] A modified SIT technology known as Release of Insects carrying a Dominant Lethal (RIDL) has been developed (Thomas et al., 2000a) and used for the control of certain insect populations. This system involves the expression of a fusion protein, tTA (tetracycline-repressible transactivator), which binds to another protein, tRe (tetracycline-responsive element), driving expression of a toxin in the absence of tetracycline. In the presence of tetracycline, the RIDL system is silenced, so that the toxin is not produced, and the insects are able to survive. To cause a reduction of the reproduction of an insect population, RIDL males are released into the environment where they mate with wild females, producing progeny that die during development in due to the absence of tetracycline in the wild. Thus, insects carrying the RIDL system can be easily mass reared with a diet supplemented with tetracycline. However, this can lead to numerous unwanted side effects, including loss of the gut microbiome, loss of symbiotic bacteria, and negative effects on mitochondrial function. Additionally, the tetracycline often does not silence completely the RIDL system, resulting in a residual toxicity that harms the males. Finally, as with SIT, three major limitations of RIDL are that males must be separated from females prior to release, releases can only be done with adults, and the effects of a RIDL intervention last only one insect generation.

[0008] In an alternative methodology to RIDL, female-specific RIDL (fsRIDL) (Labbe et al., 2012), the lethality caused by the toxin in the absence of tetracycline only affects females. This system is theoretically less effective than RIDL when comparing the total number of insects needed to be released to achieve equivalent reduction in reproduction of the target population. However, fsRIDL may be more advantageous from an economic standpoint to obtain long-term and scalable control because of three key advantages. First, as high expression of the toxin occurs only in females, males carrying the fsRIDL construct are not compromised by the damage associated to the toxin. Second, fsRIDL removes the need for sex-sorting, because females are killed off pre-release by the removal of tetracycline. Finally, releases can be performed with eggs rather than adults (since the females that hatch die during development), significantly lowering costs.

[0009] Another alternative arthropod modification strategy is the X- chromosome shredder (X-shredder) (Deredec et al., 2008; Galizi et al., 2014; Simoni et al., 2020a). In this technology, a genetic construct that is active only in males uses an endonuclease to continuously cut repetitive sequences only present in the X chromosome, damaging it in such a way that males only or predominantly pass on their non-X chromosome to the next generation, consequently generating only, or almost exclusively, males. However, such technology can in principle only be developed in species with heterogametic sex chromosomes containing repetitive sequences found exclusively on the X chromosome, thereby limiting its wide applicability to other species.

[0010] Another strategy that has been recently described is precision guided SIT (pgSIT; (Kandul et al., 2019). It is a two-system technology involving two modified insect strains. One strain carries sequences expressing two guide RNAs (gRNAs) of the CRISPR system, one targeting a genomic sequence necessary for female viability and the other a sequence necessary for male fertility, with the disruption or mutation of such sequences causing, respectively, lethality in females and sterility in males. A second insect strain contains sequences encoding a Cas9 endonuclease. When the sequences encoding the gRNAs and the Cas9 are present alone, neither exerts an effect on the carrier. However, when the Cas9 and gRNAs are present in the same individual, they interact such that the resulting progeny will consist only of sterile males and females that die during development. A major advantage of pgSIT is that the two insect strains can be independently kept and easily mass-reared, and separation of males is only explicitly needed in the generation before their release. This aspect alone makes pgSIT a cost-effective alternative because it lowers the number of insects that must be sexed by 1-2 orders of magnitude (though the sex-sorting step ahead of the release poses the same important challenges as with SIT and RIDL). Another chief advantage of pgSIT is that, as with fsRIDL, releases can be performed with eggs.

[0011] Yet another alternative, TI-pgSIT (temperature-inducible SIT; Kandul et al., 2021) was developed combining Cas9 and gRNA components of pgSIT in a single genomic locus. In this case, the single polynucleotide is placed under the control of a temperature-inducible system. Insects can be reared at low temperatures that ensure no or very limited Cas9 activity, and then reared in lab or released into the wild where the natural environmental temperature permits sufficient Cas9 activity to produce sterile males and females that die during development. Owing to this mechanism, it is not necessary to sex-sort insects prior to release, or at any point of the rearing process, and releases can be performed with eggs. However, there are added operational costs and difficulties when certain species are reared in dual temperatures, such as lack of synchronization in development due to different growth speeds at higher temperatures, or the need of ensuring a timely and harmless transfer of individuals between temperatures. Finally, as with SIT, the effects of pgSIT and TI-pgSIT interventions last only one insect generation.

[0012] Additional technologies have been described in the literature but have not yet been developed in any animal, with a notable example being the male-linked editor (MLE), also known as a Y-linked editor (YLE). In the MLE strategy, males carry on a region of the genome that is inherited only or predominantly by their male progeny (e.g., the Y chromosome in An. gambiae or the M-locus in Ae. aegypti) a transgene that causes sterility or lethality in their female progeny. Specifically, the transgene is an endonuclease that modifies genes needed for female survival or reproduction, thus disrupting their function. In an idealised system (e.g., in isolated populations), MLE males would persist in the population at the same frequency at which they were released, making this system more perduring (and thus potentially more potent) than SIT, RIDL, and their derivative strategies (Burt & Deredec, 2018). MLE genetic control strategies have great potential, but they have only recently been described on a theoretical level and none have been developed thus far. These systems too may require sex sorting and crossing of MLE males to unmodified females every generation during mass rearing to ensure the population is not eliminated through the generation of sterile or lethal traits.

[0013] A major advantage of many of these control systems, particularly those based on endonucleases, is that they could be adapted to work in any sexually reproducing animal. Indeed, CRISPR is known to be active in all kingdoms of living organisms, and all sexually reproducing species have genes that are essential for male-, female-, or both male- and female development that could be targeted to generate sterile or lethal traits useful to genetic control.

[0014] Sterile release strategies and derivatives thereof, initially developed for use in insects, are now being adapted for the control of other animal pest species (Teem et al. 2020) including invasive rodents (Manser et al. 2019; Gierus et al. 2022) and non-insect arthropods (Nuss et al. 2021).

[0015] In summary, there is still a need for cost-effective, safe, and environmentally friendly methods for biological control of pest and / or invasive animals or disease vectors such as arthropod pest species and disease vectors.

[0016] In view of the limitations of the techniques described above, the aim of the present invention is to provide a method with improved scalability for reducing the reproduction of populations of non-human, sexually reproducing, pest and / or invasive animals or disease vectors.

[0017] Within this aim, an object of the invention is to provide a method which allows easy and cost-effective mass rearing.

[0018] Another object of the invention is to provide a method which allows easy and cost-effective sex sorting.

[0019] Another object of the invention is to provide a method which allows easy and cost-effective deployment of genetically modified animals.

[0020] Yet another object of the invention is to provide a method where persistence of population suppression is controllable.

[0021] Finally, an object of the invention is to provide an eco-friendly solution for the control of pest and / or invasive animals or disease vectors.

[0022] This aim and these and other objects that will become better apparent hereinafter are achieved by a method for reducing the reproduction of a non- human, sexually reproducing, animal population comprising the steps of:

[0023] (i) providing a genetically modified male carrying:

[0024] - a first polynucleotide on the Y chromosome or on an autosome, said first polynucleotide encoding a first population suppression effector, selected from one or more of: a male and female lethality effector that causes death of the progeny of the carrier, a male and female sterility effector that causes sterility of the progeny of the carrier or causes sterility of the carrier itself, a male lethality effector that causes death of the male progeny of the carrier, a female lethality effector that causes death of the female progeny of the carrier, a female sterility effector that causes sterility of the female progeny of the carrier, a male sterility effector that causes sterility of the male progeny of the carrier or causes sterility of the carrier itself, a female-to-male sex conversion effector that causes the female progeny of the carrier to develop as a male, a male sterility and female lethality effector that causes sterility of the male progeny of the carrier or of the male carrier itself and death of the female progeny of the carrier or of the female carrier itself, and a male-sex distorter that causes the male carriers to sire a progeny which is more than 70% male; and

[0025] - a second polynucleotide on the X chromosome or on an autosome, encoding a neutralizing element configured to mitigate or remedy the effect of said first polynucleotide;

[0026] (ii) rearing the genetically modified male of step (i) allowing it to reproduce with a genetically modified female carrying the said second polynucleotide on a X chromosome or on an autosome, obtaining an animal population wherein the males carry both the first and the second polynucleotide;

[0027] (iii) removing fertile females from the population obtained in step (ii);

[0028] (iiia) optionally, rearing the males obtained in step (iii) allowing them to reproduce with a population of non -genetically modified females and isolating the male offspring carrying only the first polynucleotide;

[0029] (iv) releasing the animals obtained in step (iii) or the male offspring obtained in step (iiia) into the environment where a reduction of the reproduction of the animal is sought, obtaining a reduction of the reproduction of the animal population.

[0030] Further characteristics and advantages of the invention will become better apparent from the following detailed description and figures of which:

[0031] Figure legends

[0032] Figures 1 to 5 - Figures 1 to 5 refer to embodiments of the invention where transgenic individuals carry both a first polynucleotide (1), which causes a deleterious effect in the fitness of the animal or its progeny, and a second polynucleotide (2), which mitigates or remedies the effect of the first polynucleotide, allowing animals to be mass-reared through inbreeding. Prior to release, males carrying both polynucleotides are mated to non- genetically modified females to produce male progeny, carrying the first but not the second polynucleotide, which are then released into the environment. In these embodiments, sex sorting is not achieved by genetically encoded effectors, and so females are manually or mechanically removed from the release population.

[0033] Figure 1 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes sterility in males, or their male progeny, or their male and female progeny; and on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide. Mating of released males with wild females results in no fertile progeny if the first polynucleotide causes sterility in the released male itself, or causes sterility in its male or male and female progeny. These configurations allow for the first polynucleotide to persist in the entire male progeny for at most one generation after release.

[0034] Figure 2 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes male-specific lethality to the progeny; and, on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide. Mating of released males with wild females results in no male progeny due to the male lethality effect of the first polynucleotide. This configuration allows for the first polynucleotide to persist only during the release generation.

[0035] Figure 3 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes female lethality to the progeny; and, on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide. Mating of released males with wild females results in no female progeny due to the female lethality effect of the first polynucleotide. Male carriers are fertile and able to reproduce and this configuration leads to a perdurance of the first polynucleotide such that its female-lethal effects will remain for several generations. Figure 4 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes male and female lethality to the progeny; and, on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide. Mating of released males with wild females results in no progeny, male or female, due to the lethal effect of the first polynucleotide. This configuration allows for the first polynucleotide to persist only during the release generation.

[0036] Figure 5 illustrates the procedure from rearing to release of a transgenic strain that contains, on an autosome, a first polynucleotide that causes sterility in male progeny and lethality in female progeny; and, on the X chromosome, a second polynucleotide that mitigates or remedies for the effect of the first polynucleotide. Mating of released males with wild females results in sterile male progeny and no viable female progeny due to the male sterility and female lethality effects of the first polynucleotide. This configuration allows for the first polynucleotide to persist in part of the male progeny only on the generation after release.

[0037] Figures 6 to 12 - Figures 6 to 12 refer to embodiments of the invention where transgenic individuals carry both a first polynucleotide (1), which causes a deleterious effect in the fitness of the animal or its progeny, and an inducible second polynucleotide (2), which mitigates or remedies the effect of the first polynucleotide when in presence of the inducer, allowing animals to be mass-reared through inbreeding. The inducer is removed from the population prior to release. In some of these embodiments, females need to be manually or mechanically removed from the release population. In others, sex sorting is achieved by removing the inducer prior to release to activate a female killing or female sterilizing effector. Males carrying the first and second polynucleotides are released and mate with wild females, resulting in a reduction in reproduction.

[0038] Figure 6 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes female lethality in the progeny; and, on the X chromosome, an inducible second polynucleotide that mitigates or remedies the effect of the first polynucleotide when animals are reared with the inducer. Mating of released males with wild females results in no female progeny due to the female lethality effect of the first polynucleotide, but males inheriting it survive and are fertile. This configuration allows for the first polynucleotide to persist in the entire male progeny for several generations after release.

[0039] Figure 7 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes female sterility in the progeny; and, on the X chromosome, an inducible second polynucleotide that mitigates or remedies the effect of the first polynucleotide when animals are reared with the inducer. Female progeny are sterilized prior to release and male progeny carrying the first population suppressor are released alongside sterile females (not shown), or otherwise isolated. Mating of released males with wild females results in sterile female progeny due to the female sterile effect of the first polynucleotide, but males inheriting it survive and are fertile. This configuration allows for the first polynucleotide to persist in the entire male progeny for several generations after release.

[0040] Figure 8 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes female-to-male or male-like (intersex) conversion in the progeny; and, on the X chromosome, an inducible second polynucleotide that mitigates or remedies the effect of the first polynucleotide when animals are reared with the inducer. Prior to release, female progeny are converted and male progeny carrying the first population suppressor are released alongside intersex individuals, which are sterile (not shown), or otherwise isolated. Mating of released males with wild females results in male progeny carrying the first polynucleotide and female progeny that get converted. This configuration allows for the first polynucleotide to persist in the entire male progeny for several generations after release.

[0041] Figure 9 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes sex distortion from female to male in the female progeny; and, on the X chromosome, an inducible second polynucleotide that mitigates or remedies the effect of the first polynucleotide when animals are reared with the inducer. The inducer is removed prelease to remove females from the release population, and if sex distortion is incomplete, males must be isolated from females prior to release. Mating of released males with wild females results in a predominantly male progeny carrying the first polynucleotide. This configuration allows for the first polynucleotide to persist in the entire male progeny for several generations after release.

[0042] Figure 10 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes sterility in the male progeny or male and female progeny; and, on the X chromosome, an inducible second polynucleotide that mitigates or remedies the effect of the first polynucleotide when animals are reared with the inducer. Sterile male progeny carrying the first population suppressor are released after isolation from females prior to release. Mating of released sterile males with wild females results in no progeny, male or female. This configuration allows for the first polynucleotide to persist only during the release generation.

[0043] Figure 11 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes sterility in the male progeny and lethality in the female progeny; and, on the X chromosome, an inducible second polynucleotide that mitigates or remedies the effect of the first polynucleotide when animals are reared with the inducer. Sterile male progeny carrying the first population suppressor are produced and then released. Mating of released sterile males with wild females results in no progeny, male or female. This configuration allows for the first polynucleotide to persist only during the release generation.

[0044] Figure 12 illustrates the procedure from rearing to release of a transgenic strain that contains, on an autosome, a first polynucleotide that causes sterility in the male progeny and lethality in the female progeny; and, on the X chromosome, an inducible second polynucleotide that mitigates or remedies the effect of the first polynucleotide when animals are reared with the inducer. Sterile male progeny carrying the first population suppressor are produced and then released. Mating of released sterile males with wild females results in no progeny, male or female. This configuration allows for the first polynucleotide to persist only during the release generation.

[0045] Figures 13 to 22 - Figures 13 to 22 refer to embodiments of the invention where transgenic individuals are mass-reared because they carry both a first polynucleotide, which causes a deleterious effect in the fitness of the individual, a second polynucleotide, which mitigates or remedies the effect of the first polynucleotide, and, genetically linked to the second, a repressible third polynucleotide, which encodes a female lethality effector that is inhibited in presence of the repressor. Animals are mass-reared through inbreeding in the presence of the repressor. In these figures and embodiments, sex sorting is achieved prior to release by removing the repressor during rearing, thus activating the third polynucleotide that causes lethality in females. Depending on when the repressor is removed, and at what developmental stage the female lethal effector must be active to induce female lethality, females may be removed in the generation in which the repressor is removed (not shown), or in the subsequent generation. Males containing all three polynucleotides are released into the environment and mate with wild females, resulting in a reduction in reproduction.

[0046] Figure 13 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes female sterility in the progeny; on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide and a repressible third polynucleotide, genetically linked to the second, that causes female lethality and is inhibited when the carrier is reared with the repressor. Female progeny of released males are killed by the third polynucleotide, thus removing simultaneously the second and third polynucleotides from the population. Male progeny of released males only inherit the first polynucleotide leading to sterility in their female progeny but allowing the production of fertile male progeny. This configuration allows for the first polynucleotide to persist in the entire male progeny for several generations after release.

[0047] Figure 14 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes female lethality in the progeny; on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide and a repressible third polynucleotide, genetically linked to the second, that causes female lethality and is inhibited when the carrier is reared with the repressor. Female progeny of released males are killed by the third polynucleotide, thus removing simultaneously the second and third polynucleotides from the population. Male progeny of released males only inherit the first polynucleotide leading to lethality in their female progeny but allowing the production of fertile male progeny. This configuration allows for the first polynucleotide to persist in the entire male progeny for several generations after release.

[0048] Figure 15 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes male-specific sterility in the progeny; on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide and a repressible third polynucleotide, genetically linked to the second, that causes female lethality and is inhibited when the carrier is reared with the repressor. Female progeny of released males is killed by the third polynucleotide, thus removing simultaneously the second and third polynucleotides from the population. Male progeny of released males only inherit the first polynucleotide leading to sterility in their male progeny. This configuration allows for the first polynucleotide to persist in the male progeny for two generations after release.

[0049] Figure 16 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes male sterility; on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide and a repressible third polynucleotide, genetically linked to the second, that causes female-specific lethality and is inhibited when the carrier is reared with the repressor. Female progeny of released males are killed by the third polynucleotide, thus removing simultaneously the second and third polynucleotides from the population. Male progeny of released males only inherit the first polynucleotide leading to their sterility. This configuration allows for the first polynucleotide to persist in the entire male progeny for one generation after release.

[0050] Figure 17 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes lethality in the male progeny, or lethality in the male and female progeny; on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide and a repressible third polynucleotide, genetically linked to the second, that causes female lethality and is inhibited when the carrier is reared with the repressor. Female progeny of released males is killed by the third polynucleotide, thus removing simultaneously the second and third polynucleotides from the population. Male progeny of released males inherit only the first polynucleotide leading to lethality in their male progeny or in male and female progeny. This configuration allows for the first polynucleotide to persist in the entire male progeny for one generation after release.

[0051] Figure 18 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes male-specific lethality; on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide and a repressible third polynucleotide, genetically linked to the second, that causes female lethality and is inhibited when the carrier is reared with the repressor. Female progeny of released males are killed by the third polynucleotide, thus removing simultaneously the second and third polynucleotides from the population. Male progeny of released males inherit only the first polynucleotide leading to their lethality. This configuration allows for the first polynucleotide to be removed from the population in the release generation.

[0052] Figure 19 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes sterility in the male progeny and lethality in the female progeny; on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide and a repressible third polynucleotide, genetically linked to the second, that causes female-specific lethality and is inhibited when the carrier is reared with the repressor. Female progeny of released males are killed by the third polynucleotide, thus removing simultaneously the second and third polynucleotides from the population. Male progeny of released males inherit only the first polynucleotide leading to sterility and lethality in their male and female progeny, respectively. This configuration allows for the first polynucleotide to persist in the entire male progeny for two generations after release.

[0053] Figure 20 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes female-to-male or male-like (intersex) conversion in the progeny; on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide and a repressible third polynucleotide that causes female-specific lethality and is inhibited when the carrier is reared with the repressor. Female progeny of released males are killed by the third polynucleotide, thus removing simultaneously the second and third polynucleotides from the population. Male progeny of released males inherit only the first polynucleotide leading to the conversion of their female progeny to sterile males, but producing fertile male progeny. This configuration allows for the first polynucleotide to persist in the entire male progeny for several generations after release.

[0054] Figure 21 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes lethality in the female progeny; on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide and a repressible third polynucleotide, genetically linked to the second, that causes female-specific sterility or male and female sterility and is inhibited when the carrier is reared with the repressor. When the third polynucleotide causes male and female sterility, males isolated pre-release will be sterile and, when released, will produce no progeny. If instead the third polynucleotide causes female-specific sterility, released males are fertile and produce a sterile female progeny through the effect of the third polynucleotide. Male progeny of released males inherit the first polynucleotide leading to lethality in their female progeny but keep producing fertile male progeny. This allows for the first polynucleotide to persist in the entire male progeny for several generations after release.

[0055] Figure 22 illustrates the procedure from rearing to release of a transgenic strain that contains, on the Y chromosome, a first polynucleotide that causes lethality in the female progeny; on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide and a repressible third polynucleotide, genetically linked to the second, that causes female-to-male or male-like sterile individuals and is inhibited when the carrier is reared with the repressor. Female progeny of released males are converted to male- or sterile male-like-progeny by the third polynucleotide, thus removing simultaneously the second and third polynucleotides from the population. Male progeny of released males inherit the first polynucleotide leading to lethality in their female progeny but producing fertile male progeny. This configuration allows for the first polynucleotide to persist in the entire male progeny for several generations after release.

[0056] Figure 23 illustrates the procedure from rearing to release of a transgenic strain that contains, on an autosome, a first polynucleotide that causes lethality in the female progeny and can copy itself to the homologous chromosome; on the X chromosome, an inducible second polynucleotide that mitigates or remedies the effect of the first polynucleotide when carriers are reared in the presence of an inducer. The inducer is removed from the population prior to release, thus preventing mitigation or remedy of the effect of the first polynucleotide so that female progeny are killed but male progeny carrying the first population suppressor are isolated for release. Mating of released males with wild females results in non-viable female progeny and male progeny that carry the first polynucleotide. Thus, the first polynucleotide persists and spreads over multiple generations through the males, generating continued female lethal effects in absence of the inducer. This configuration allows for the first polynucleotide to persist and increase in frequency in the entire male progeny for several generations after release.

[0057] Figure 24 illustrates the procedure from rearing to release of a transgenic strain that contains, genetically linked in the same autosome, a first polynucleotide that causes sterility in the male progeny and lethality in the female progeny and an inducible second polynucleotide that mitigates or remedies the effect of the first polynucleotide when carriers are reared in the presence of the inducer. The inducer is removed from the population prior to release, thus preventing mitigation or remedy of the effect of the first polynucleotide, such that female progeny are killed but sterile male progeny carrying the first population suppressor survive and are released. Mating of released males with wild females results in no progeny, male or female.

[0058] Figure 25 illustrates the procedure from rearing to release of a transgenic strain that contains, genetically linked in the same autosome, a first polynucleotide that causes sterility in the male progeny and lethality in the female progeny and an inducible second polynucleotide that mitigates or remedies the effect of the first polynucleotide in the presence of the inducer. Sex sorting is required to separate males from females prior to release. The inducer is removed naturally from the population during release, thus preventing mitigation or remedy of the effect of the first polynucleotide. Female progeny are killed but sterile male progeny carrying the active first population suppressor survive. Mating of said sterile male progeny with wild females results in no progeny, male or female.

[0059] Figure 26 illustrates the procedure from rearing to release of a transgenic strain that contains, genetically linked in the same autosome, a first polynucleotide that causes sterility in the male progeny and lethality in the female progeny; and a repressible second polynucleotide that mitigates or remedies the effect of the first polynucleotide when the carriers are reared in absence of the repressor, thus being active when the carrier is reared in a regular diet. The repressor is administered to the population prior to release, thus preventing mitigation or remedy of the effect of the first polynucleotide so that female progeny die and sterile male progeny carrying the first population suppressor survive and are isolated for release. Mating of released sterile males with wild females results in no progeny, male or female.

[0060] Figure 27 illustrates the procedure from rearing to release of a transgenic strain that contains, on an autosome, a first polynucleotide that causes sterility in male progeny and lethality in female progeny; on the X chromosome, a second polynucleotide that mitigates or remedies the effect of the first polynucleotide and a repressible third polynucleotide, genetically linked to the second, that encodes a female-specific lethality effector that is inhibited when in presence of the repressor. Carriers of all polynucleotides can be reared at scale through inbreeding in the presence of a repressor. Sex sorting is achieved by removing the repressor during the pre-release generation. Mating of released males containing the three polynucleotides results in lethality in the female progeny due to the action of the third polynucleotide. Males carrying the first polynucleotide mate with wild females, resulting in sterile male progeny and no viable female progeny. This configuration allows for the first polynucleotide to persist in part of the male progeny for two generations after release.

[0061] The present invention is generally applicable to all non-human animals that reproduce sexually. In particular, this invention lends itself well to diploid or haplodiploid pests and / or invasive species or vectors of disease wherein males and females are sexually dimorphic.

[0062] The first step of the method according to the invention consists in providing a genetically modified male carrying a first polynucleotide on the Y chromosome or on an autosome, said first polynucleotide encoding a first population suppression effector, and a second polynucleotide on the X chromosome or on an autosome, thus in a region of the genome that is inherited by the female progeny of said genetically modified males, said second polynucleotide encoding a neutralizing element configured to mitigate or remedy the effect of said first population suppression effector;

[0063] The first population suppression effector is selected from one or more of: a male and female lethality effector that causes death of the progeny of the carrier (Figure 4), a male and female sterility effector that causes sterility of the progeny of the carrier or causes sterility of the carrier itself (Figure 1), a male lethality effector that causes death of the male progeny of the carrier (Figure 2), a female lethality effector that causes death of the female progeny of the carrier (Figure 3), a female sterility effector that causes sterility of the female progeny of the carrier (Figure 1), a male sterility effector that causes sterility of the male progeny of the carrier or causes sterility of the carrier itself (Figure 1), a female-to-male sex conversion effector that causes a female progeny of the carrier to develop as a male, a male sterility and female lethality effector that causes sterility of the male progeny of the carrier or of the male carrier itself and death of the female progeny of the carrier or of the female carrier itself (Figure 6), and a malesex distorter that causes the male carriers to sire a progeny which is more than 70% male (Figure 9).

[0064] Within the scope of the present invention the term “lethality effector” indicates an effector causing death of at least 40% of the target progeny of the carrier, preferably at least 70% of the target progeny of the carrier, more preferably at least 95% of the target progeny of the carrier.

[0065] Within the scope of the present invention the term “sterility effector” indicates an effector causing at least 40% sterility in the target progeny of the carrier or in the carrier itself, preferably at least 70% sterility in the target progeny of the carrier or in the carrier itself, more preferably at least 95% sterility in the target progeny of the carrier or in the carrier itself.

[0066] Within the scope of the present invention “target progeny” means a progeny of a sex affected by the population suppression effector, being male, female or both.

[0067] In the second step of the method, the genetically modified male obtained in step (i) is reared with a genetically modified female carrying the said second polynucleotide on a X chromosome or on an autosome (Figures 1-5). In this way, the neutralizing element prevents, or compensates for, the effect of the first polynucleotide resulting in the carrier being able to survive and reproduce with similar fitness to non-genetically modified individuals thus obtaining an animal population wherein the males carry both the first and the second polynucleotide. In some embodiments, the second polynucleotide is on an autosome at a locus that enables no more than 60% genetic linkage with the first polynucleotide. In some preferred embodiments the first polynucleotide and the second polynucleotide are on different autosomes.

[0068] In some embodiments, the second polynucleotide is on the X chromosome of males, or both males and females (Figures 1-5). This allows the neutralizing element to segregate into the female progeny but not into the male progeny of a male carrier. In such embodiments, the first polynucleotide can be located on an autosome and be transmitted to male progeny without transferring the neutralizing element (Figure 5 pre-release). In preferred embodiments, the first polynucleotide is homozygous on the autosomes of the male carrier such that it is transferred to all of the progeny. In other preferred embodiments, the first polynucleotide is on the Y chromosome so that males transmit the first polynucleotide to all of their male progeny, also in this case without transmitting the neutralizing element (Figures 1-4).

[0069] In some embodiments, the second polynucleotide is on an autosome (Figures 24 and 25). This allows the said second polynucleotide encoding the neutralizing element to be inherited by male and female progeny. In preferred embodiments, the second polynucleotide is inserted on an autosome within a sex-related region that is preferentially inherited by the female progeny of male carriers, such as the non-male determining m-locus in animal species wherein sex is determined by a male determining M-locus, so that the second polynucleotide is inherited preferentially by the female progeny.

[0070] The third step of the method (iii) is the removal of fertile females from the population obtained in step (ii). Preferably, at least 50% of fertile females are removed from the population, or in a preferred embodiment at least 95% of fertile females are removed from the population, most preferably at least 99% of fertile females are removed from the population. Removal of females can be obtained in different ways. In some embodiments, such isolation can occur by mechanical removal, such as the sex sorting of the females from the population reared in step (ii) that is image-based (Bouyer et al., 2022; Crawford et al., 2020), size-based (Carvalho et al., 2014), or fluorescence-based (Marois et al., 2012)).

[0071] Finally, in step (iv) the animals obtained in step (iii) or the male offspring obtained in step (iiia) are released into the environment where a reduction of the reproduction of the animal is sought, obtaining a reduction of the reproduction of the animal population.

[0072] The method of the present invention may optionally comprise a step (iiia) of mating males obtained in step (iii) with a population of non- genetically modified females and isolating the male progeny carrying only the first polynucleotide (Figures 1-5).

[0073] In some embodiments, males carrying the first polynucleotide can be outcrossed to females that do not carry the neutralizing element immediately prior to release, as in step (iiia), to isolate males that do not carry the neutralizing element. This step is optional as released males carrying both first and second polynucleotides will produce a first- or second-generation male progeny that do not carry the second polynucleotide, a feature that, in some embodiments, is preferred.

[0074] According to a preferred embodiment of the invention, the second polynucleotide encodes a neutralizing element that is inducible through an inducer and rearing in step (iiib) is conducted in presence of said inducer which activates such inducible neutralizing element (Figures 6-12 and 23- 25). The inducible neutralizing element is not activated by the natural environment where a reduction in the reproduction of the animal pest population is sought. In fact, the inducer is either an uncommon condition in the natural environment where the reduction of reproduction is sought, or a compound which, appropriately, is never or only rarely present in the same natural environment in sufficient abundance or concentration to induce the neutralizing element.

[0075] In an embodiment of the method of the present invention (Figures 24 and 25):

[0076] - the first polynucleotide is on an autosome;

[0077] - the second polynucleotide encodes an inducible neutralizing element;

[0078] - preferably, the first and second polynucleotide are genetically linked within the same genomic locus, or are within one centimorgan of each other, meaning that they are inherited together at least 99% of the time; and

[0079] - rearing in step (ii) is conducted in presence of an inducer activating the inducible neutralizing element.

[0080] In an embodiment of the method of the present invention:

[0081] - the first population suppression effector is selected from a female lethality effector (Figure 6), a female sterility effector (Figure 7), a female- to-male sex conversion effector (Figure 8), and a male-sex distorter (Figure 9), a male and female sterility effector (Figure 10), and a male sterility and female lethality effector (Figure 11); preferably:

[0082] - the first polynucleotide encoding the first population suppression effector is on an autosome (Figure 12);

[0083] - the neutralizing element encoded by the second polynucleotide is an inducible neutralizing element; preferably the first and second polynucleotides are both on an autosome, genetically linked within the same genomic locus, or are within one centimorgan of each other (Figures 24 and 25).

[0084] - rearing in step (ii) is conducted in presence of an inducer activating the inducible neutralizing element;

[0085] - removing fertile females in step (iii) is obtained by removing the inducer, thus activating the first population suppression effector. In absence of the inducer, the neutralizing element becomes inactive and thus it no longer prevents or compensates for the effects caused by the first polynucleotide (Figures 6-12, 23 and 24). In case of, for example, a first polynucleotide encoding a female lethality effector and a male sterility and female lethality effector, and a second polynucleotide encoding the inducible neutralizing element, females can be removed from the population by removal of the inducer. As inducers can be small molecules supplemented in the animal diet, for example tetracycline or tetracyclineanalog inducers wherein the neutralizing element may be placed under control of a tet-on system (Lycett et al. 2004) (J. Chen et al., 2021), female lethality can be prevented by supplementing the drug within the diet.

[0086] In some embodiments, the neutralizing element is under the control of an inducible promoter, for example SEQ ID NO:41, selected from a temperature-inducible promoter that can increase or decrease gene expression in response to changes in temperature , a drug-inducible promoter that can increase gene expression in response to a drug or chemical (J. Chen et al., 2021), or a transactivator-inducible promoter that can increase gene expression in response to a genetically encoded transactivator or transcription factor (Schetelig et al., 2022).

[0087] In an embodiment of the method of the present invention (Figure 26):

[0088] - the first polynucleotide encodes a male sterility and female lethality effector;

[0089] - the neutralizing element encoded by the second polynucleotide is a repressible neutralizing element, for example, SEQ ID NO: 70; preferably the first and second polynucleotides are both on an autosome, genetically linked within the same genomic locus, or are within one centimorgan of each other;

[0090] - rearing in step (ii) is conducted in absence of a repressor thus activating the repressible neutralizing element;

[0091] - removing fertile females in step (iii) is obtained by adding said repressor, thus activating the first population suppression effector. In presence of said repressor, the neutralizing element becomes inactive and thus it no longer prevents or compensates for the effects caused by the first polynucleotide. Thus, females can be removed from the population in the presence of said repressor. As repressors can be small molecules supplemented in the animal diet, for example tetracycline or tetracyclineanalog repressors wherein the neutralizing element may be placed under control of a tet-off system (Lycett et al. 2004), female lethality can be prevented by rearing the animals on a diet that does not contain said repressors.

[0092] In an embodiment of the invention as described above in any of the previous embodiments:

[0093] - the genetically modified female of step (ii) additionally carries on an X chromosome or on an autosome a third polynucleotide encoding a second population suppression effector that is a female population suppression effector that is repressible through a repressor, for example, SEQ ID NOs:66, 67, 70 (Figures 13-22) selected from a female lethality effector that, in absence of said repressor, causes death of the female carrier (Figures 13-20), a female sterility effector that, in absence of said repressor, causes sterility of the female carrier (Figure 21), a female-to-male sex conversion effector that, in absence of said repressor, causes sex conversion of female carriers into males (Figure 22), a male and female sterility effector that, in absence of said repressor (Figure 21), causes sterility of the progeny of the carrier or causes sterility of the carrier itself, and a male sterility and female lethality effector that, in absence of said repressor, causes sterility of the male progeny of the carrier or of the male carrier itself and death of the female progeny of the carrier or of the female carrier itself (Figure 27);

[0094] - rearing in step (ii) is conducted in presence of a repressor inactivating the third polynucleotide; and

[0095] - removing fertile females in step (iii) is obtained by removing the repressor, thus activating the third polynucleotide.

[0096] In some embodiments, the third polynucleotide encodes an exogenous toxin, a pro-apoptotic gene, or a dominant effector impairing development. The above described third polynucleotide encodes a female-specific population suppression effector (Figure 13-20, 27). In this method, toxins, pro-apoptotic genes, or dominant effectors are placed under control of a female-specific expression system that causes lethality or impairs development only of females. Such a system could allow females to be killed at any time by removal of the repressor, if the lethal effector affects all developmental stages, for example certain toxins and proapoptotic genes. Alternatively, the system may be specific to a developmental stage so that fertile females are removed only if the repressor is absent during the developmental stage at which the effector must be active; for example, disruption of the function of a gene required for female-specific embryo development will only affect females reared in absence of the repressor at the embryo stage, but not in later stages. Examples of exogenous toxins include Bacillus thuringiensis toxin and its derivatives (Bti, Bsp, etc.), Binary (BinA / B) toxin (Sharma & Kumar, 2022), Clostridium-derived toxins such as PMP1 (Contreras et al., 2019), snake toxic or neurotoxic phospholipase A2 enzymes, cobrotoxin, saxitoxin, microcystin and scorpion insect sodium channel toxin (Turell & Middlebrook, 1988), among others. Examples of pro-apoptotic genes include hid, reaper, and grim (Goyal et al., 2000), Michelob x (mx) (Zhou et al., 2005), bax (Yamamoto et al., 2019), among others. Examples of other dominant effectors include an fsRIDL construct such as a tetracycline-controlled transactivator protein, dominant negative mutations in and around the female-specific exon of the doublesex gene (Yadav et al., 2023a) (SEQ ID NOs:45-47, 76), a masculinising gene such as yobl and nix (SEQ ID NOs:63-65), expression of a targeted genome-editing system designed to create null mutations in female or female and male essential genes (SEQ ID NOs: 24-29, combined with SEQ ID NOs:30-32 and SEQ ID NOs:42-65), among others.

[0097] In some embodiments, the female-specific expression system is selected from a female-specific promoter, a female-specific splice factor, a female-specific binary expression system, and a female-specific inducible expression system.

[0098] In a preferred embodiment of the invention:

[0099] - the second polynucleotide of steps (i) and (ii) is on an autosome;

[0100] - the genetically modified female of step (ii) additionally carries on an autosome a third polynucleotide encoding a second population suppression effector that is a female population suppression effector that is repressible through a repressor, (e.g. SEQ ID NOs:66, 67, 70), said female population suppression effector being selected from a female lethality effector that, in absence of said repressor, causes death of the female carrier, a female sterility effector that, in absence of said repressor, causes sterility of the female carrier, a female-to-male sex conversion effector that, in absence of said repressor, causes sex conversion of female carriers into males, a male and female sterility effector that, in absence of said repressor, causes sterility of the progeny of the carrier or causes sterility of the carrier itself, and a male sterility and female lethality effector that, in absence of said repressor, causes sterility of the male progeny of the carrier or of the male carrier itself and death of the female progeny of the carrier or of the female carrier itself;

[0101] - the second and the third polynucleotides are both on an autosome, genetically linked within the same genomic locus, or are within one centimorgan of each other;

[0102] - rearing in step (ii) is conducted in presence of said repressor inactivating the third polynucleotide; and

[0103] - removing fertile females in step (iii) is obtained by removing the repressor, thus activating the second population suppression effector.

[0104] In an embodiment of the invention:

[0105] - the first polynucleotide encodes a population suppression effector selected from: a) a female sterility effector (Figure 13), a female lethality effector (Figure 14), a male-sex distorter, and a female-to-male sex conversion effector (Figure 20); and b) a male sterility effector (Figures 15 and 16), a male and female lethality effector (Figure 17), and a male sterility and female lethality effector (Figure 19);

[0106] - the genetically modified female of step (ii) additionally carries on an X chromosome or on an autosome a third polynucleotide encoding a second population suppression effector that is a female population suppression effector that is repressible through a repressor, for example SEQ ID NOs:66, 67, 70, said female population suppression effector being selected from a female lethality effector that, in absence of said repressor, causes death of any female carrier (Figures 13-20), a female sterility effector that, in absence of said repressor, causes sterility of the female carrier (Figure 21), a female-to-male sex conversion effector that, in absence of said repressor, causes sex conversion of female carriers into males (Figure 22), a male and female sterility effector that, in absence of said repressor, causes sterility of the progeny of the carrier or causes sterility of the carrier itself (Figure 21), and a male sterility and female lethality effector that, in absence of said repressor, causes sterility of the male progeny of the carrier or of the male carrier itself and death of the female progeny of the carrier or of the female carrier itself;

[0107] - rearing in step (ii) is conducted in presence of said repressor inactivating the second population suppression effector; and

[0108] - removing fertile females in step (iii) is obtained by removing said repressor, thus activating the second population suppression effector.

[0109] In another embodiment of the invention:

[0110] - The first polynucleotide encodes a population suppression effector selected from: a) a female sterility effector (Figure 13), a female lethality effector (Figure 14), a male-sex distorter, and a female-to-male sex conversion effector (Figure 20); and b) a male sterility effector (Figures 15 and 16), a male and female lethality effector (Figure 17), and a male sterility and female lethality effector (Figure 19);

[0111] - the second polynucleotide of steps (i) and (ii) is on an autosome or on an X chromosome;

[0112] - the genetically modified female of step (ii) additionally carries, on an autosome or on an X chromosome, a third polynucleotide encoding a second population suppression effector that is a female population suppression effector that is repressible through a repressor, for example SEQ ID NOs:66, 67, 70, said female population suppression effector being selected from a female lethality effector that, in absence of said repressor, causes death of the female carrier (Figures 13-20), a female sterility effector that, in absence of said repressor, causes sterility of the female carrier (Figure 21), a female-to-male sex conversion effector that, in absence of said repressor, causes sex conversion of female carriers into males (Figure 22), a male and female sterility effector that, in absence of said repressor, causes sterility of the progeny of the carrier or causes sterility of the carrier itself (Figure 21), and a male sterility and female lethality effector that, in absence of said repressor, causes sterility of the male progeny of the carrier or of the male carrier itself and death of the female progeny of the carrier or of the female carrier itself (Figure 26);

[0113] - the second and third polynucleotides are on the same chromosome, genetically linked within the same genomic locus, or are within one centimorgan of each other (Figures 13-22);

[0114] - rearing in step (ii) is conducted in presence of a repressor inactivating the second population suppression effector; and

[0115] - removing fertile females in step (iii) is obtained by removing the repressor, thus activating the second population suppression effector. In practice, the animals are reared during the entirety of their life cycle, or only during specific life stages, in the presence of a small molecule repressor that binds to the specific transactivator (blocking its function) in charge of female killing, or sterility, and thus also enabling survival of fertile females. When the repressor is removed from the diet, the transactivator becomes active and promotes female killing or sterility, irrespective of the effector located on the first polynucleotide.

[0116] In another embodiment of the invention (Figures 15-18):

[0117] - The first polynucleotide encodes a population suppression effector selected from a male sterility effector (Figures 15 and 16), and a male and female lethality effector (Figure 17 and 18);

[0118] - the second polynucleotide of steps (i) and (ii) is on the X- chromosome, or on a sex-related region of an autosome that is almost exclusively inherited by the female progeny of a male carrier (such as the non-male determining m-locus in animals for which sex is determined by a male-determining M-locus);

[0119] - the genetically modified female of step (ii) additionally carries, on an autosome or on an X chromosome, a third polynucleotide encoding a second population suppression effector that is a female population suppression effector that is repressible (e.g. SEQ ID NOs:66, 67, 70) through a repressor, said female population suppression effector being selected from a female lethality effector that, in absence of said repressor, causes death of the female carrier (Figures 13-20), and a female sterility effector that, in absence of the repressor, causes sterility of the female carrier;

[0120] - rearing in step (ii) is conducted in presence of a repressor inactivating the second population suppression effector; and

[0121] - removing fertile females in step (iii) is obtained by removing the repressor, thus activating the second population suppression effector.

[0122] This embodiment can generate a reduction in the reproduction of an animal population for precisely two animal generations if the first polynucleotide generates complete sterility of male carriers, or complete lethality in the progeny of male carriers, and if the third polynucleotide causes complete lethality of female carriers, or complete sterility of female carriers. In this way, the female progeny of released males will be killed or rendered sterile by virtue of the third polynucleotide that is activated in the absence of a repressor. Released males are fertile by virtue of the activity of the second polynucleotide. Released males mate with wild females and produce male progeny (“second generation males”) that carry the first polynucleotide, but do not carry the second polynucleotide that is inherited by the female but not male progeny. Accordingly, second generation males are sterile, or unable to produce viable progeny. These features, combined, result in a reduction in the reproduction of the animal population for two animal generations through the generation, in the progeny of released males, of 1) sterile or in viable females and 2) sterile males that can reduce the fertility of wild females. In a preferred embodiment, the first polynucleotide is on the Y chromosome or on an autosome and homozygous in released males, thus, all male progeny will inherit the active first polynucleotide encoding a population suppression effector (Figures 13-27).

[0123] In an embodiment of the invention as described above in any of its embodiments, the first polynucleotide encodes a construct on an autosome capable of homing in the germline (Figure 23). “Homing” signifies that the construct copies itself on the homologous genomic locus located on the homologous chromosome through recombinatorial repair (homology- directed repair).

[0124] In a further embodiment, the first polynucleotide encodes a construct on an autosome capable of homing in the germline and somatic tissues.

[0125] In an embodiment of the invention as described above in any of its embodiments (Figures 1-27), one or both of the first and the third polynucleotides are selected from a targeted genome-editing system that creates one or more DNA sequence mutations, which disrupt the function of one or more endogenous essential genes or sex-specific splice variants thereof, and an RNA interference system (RNAi) that disrupts the function of one or more endogenous essential genes or sex-specific splice variants thereof.

[0126] In an embodiment of the invention as described above in any of its embodiments (Figures 1-27), one or both of the first and the third polynucleotides are selected from:

[0127] - a CRISPR system, preferably selected from a CRISPR-Cas9 system and a CRISPR-Casl2a system, and

[0128] - an RNA interference (RNAi) system.

[0129] In an embodiment of the invention as described above in any of its embodiments (Figures 1-27), the first polynucleotide encodes a CRISPR system and the second polynucleotide encodes one of an anti-CRISPR protein, an anti-CRISPR RNA, an RNAi targeting the CRISPR system, and a recoded copy of a gene inactivated by the first polynucleotide. In a preferred embodiment, the second polynucleotide encodes AcrIIA4. However, the second polynucleotide is not constrained to AcrIIA4, as it can encode any other additional molecule that acts as anti-CRISPR, such as AcrE(l-4), AcrF(l-lO), AcrIIA(l-3), AcrIIC(l-3), among others.

[0130] In a preferred embodiment of the invention, the non-human, sexually reproducing, animal is an invertebrate, preferentially selected from a pest and / or invasive arthropod species and vector of disease.

[0131] To obtain the genetically modified animals that encode the first polynucleotide in their genome, or the second polynucleotide, or the third polynucleotide, or any combination thereof, these polynucleotides need to be inserted into the animal’s genome by germline transformation. Each polynucleotide can be delivered into, or created within, germline cells or tissues using a gene delivery method (Terradas et al., 2022) that includes embryo microinjection that has been widely applied in animal pest and / or invasive species or disease vector species (e.g. SEQ ID Nos:36, 37), adult injection, vector-mediated gene transfer techniques such as viral infection / transfection, or protein- or lipid-based gene delivery techniques delivering “naked” polynucleotides or nucleic acid structures (e.g., electroporation or gene gun). In some preferred embodiments the genetically modified animal is first modified with the second polynucleotide and then secondarily modified with the first polynucleotide, thus, ensuring the effect of the first polynucleotide is mitigated or remedied at the point of creation.

[0132] In some embodiments the first polynucleotide encodes a first population suppression effector, selected from one or more of: a male and female lethality effector that causes death of at least 50% of the progeny of the carrier, or in a preferred embodiment at least 95% of the progeny of the carrier, a male and female sterility effector that causes at least 50% sterility of the progeny of the carrier or causes at least 50% sterility of the carrier itself, or in a preferred embodiment at least 95% sterility of the progeny of the carrier or causes at least 95% sterility of the carrier itself, a male lethality effector that causes death of at least 50% of the male progeny of the carrier, or in a preferred embodiment at least 95% of the male progeny of the carrier, a female lethality effector that causes death of at least 50% of the female progeny of the carrier, or in a preferred embodiment at least 95% of the female progeny of the carrier, a female sterility effector that causes at least 50% sterility of the female progeny of the carrier, or in a preferred embodiment at least 95% sterility of the female progeny of the carrier, a male sterility effector that causes at least 50% sterility of the male progeny of the carrier or causes at least 50% sterility of the carrier itself, or in a preferred embodiment at least 95% sterility of the male progeny of the carrier or causes at least 95% sterility of the carrier itself, a female-to-male sex conversion effector that causes at least 50% of the female progeny of the carrier to develop as a male, or in a preferred embodiment at least 95% of the female progeny of the carrier to develop as a male, a male sterility and female lethality effector that causes at least 50% sterility of the male progeny of the carrier or of the male carrier itself and death of at least 50% of the female progeny of the carrier or of the female carrier itself, or in a preferred embodiment at least 95% sterility of the male progeny of the carrier or of the male carrier itself and death of at least 95% of the female progeny of the carrier or of the female carrier itself and a male-sex distorter that causes the male carriers to sire a progeny which is more than 70% male, or in a preferred embodiment at least 95% male.

[0133] In some embodiments, the first polynucleotide encodes one or more effectors selected from: a null mutation within an endogenous essential gene, a male-determining factor that promotes male sexual development, a toxin that causes death of a cell, tissue or animal, a toxin that specifically causes death of female cells, female tissue or the females, an RNA interference system (RNAi) that disrupts the function of one or more endogenous essential genes, a targeted genome-editing system that creates one or more mutations that disrupt the function of one or more endogenous essential genes, an X-shredder, and a targeted epigenome-editing system that disrupts the function of one or more endogenous essential genes.

[0134] In some embodiments, the endogenous essential gene has an essential role such as male fertility, female fertility, male and female fertility, male viability, female viability, male and female viability, or female sex determination.

[0135] In some embodiments, the first polynucleotide encodes a null mutation, and the second polynucleotide encodes a functional copy of the endogenous essential gene. Such functional copies could be created using a targeted genome-editing system by, for example, allowing the creation of end-joining mutations without the introduction of foreign DNA, or through the introduction of a specific mutation by homology-directed repair, prime editing, or base editing.

[0136] In some embodiments, the first polynucleotide encodes one amongst an RNAi, a targeted genome-editing system, and a targeted epigenomeediting system, resulting in the loss of function of an endogenous essential gene, while the second polynucleotide encodes a recoded copy of said endogenous essential gene that cannot be inactivated by the product of the first polynucleotide. In cases where multiple endogenous essential genes are targeted, the second polynucleotide encodes a recoded copy of each endogenous essential gene that cannot be targeted by the first polynucleotide.

[0137] In some embodiments, the first polynucleotide comprises an RNAi system selected from a microRNA (miRNA), a short-hairpin RNA (shRNA), a piwi-interacting RNA (piRNA), and a CRISPR-interference system (CRISPRi).

[0138] In some embodiments, the first polynucleotide or third polynucleotide encode a male-determining factor, including for example, Guyl (Criscione et al., 2016), nix (Hall et al., 2015; P. Liu et al., 2020), yobl (Krzywinska et al., 2016), moy (Meccariello et al., 2019), or the doublesex male-splice variant (dsxm) (Dauwalder et al., 2002; Nothiger et al., 1987).

[0139] In some embodiments, the targeted genome-editing system is selected from an RNA-guided DNA endonuclease, a DNA-guided DNA endonuclease, an RNA-guided nickase, a DNA-guided nickase, an RNA- guided base editor (Anzalone et al., 2020), an RNA-guided prime editor (Anzalone et al., 2020), an RNA-guided cleavage free editor, an RNA- guided transposase or integrase, a zinc-finger nuclease (ZFN), a homing endonuclease or meganuclease, and a Transcription Activator-Like Effector Nuclease (TALEN)(Shamshirgaran et al., 2022).

[0140] In some embodiments, the RNA-guided endonuclease is a CRISPR system based on Cas9 or a variant thereof. Examples of Cas9 variants include high-fidelity Cas9 variants, such as eSpCas9(l.l), Cas9-HF1, HypaCas9, Cas9_R63A / Q768A, evoCas9, HiFi Cas9, Sniper-Cas9, and Sniper2L, and Cas9 PAM variants. In some embodiments, the first polynucleotide comprises an RNA- guided population suppression effector selected from a CRISPRi, an RNA- guided transcriptional activator, an RNA-guided endonuclease, an RNA- guided nickase, an RNA-guided base editor, an RNA-guided prime editor, an RNA-guided cleavage free editor, an RNA-guided RNA editor, and an RNA-guided transposase or integrase.

[0141] In some embodiments, the RNA-guided population suppression effector is a CRISPR-based population suppression effector selected from a CRISPRi, a CRISPR-based RNA-guided transcriptional activator (CRISPRa) population suppression effector, a CRISPR-based RNA-guided DNA endonuclease population suppression effector, a CRISPR-based RNA- guided nickase population suppression effector, a CRISPR-based RNA- guided base editor population suppression effector, a CRISPR-based RNA- guided prime editor population suppression effector, a CRISPR-based RNA- guided cleavage free editor population suppression effector, and a CRISPR- based RNA-guided transposase or integrase population suppression effector.

[0142] In some embodiments, the first polynucleotide encodes a CRISPR- based population suppression effector and the second polynucleotide encodes an anti-CRISPR protein or RNA (SEQ ID NO: 33) that can prevent or reduce CRISPR activity, or an RNAi targeting Cas9 or the associated gRNA.

[0143] In some embodiments, the second polynucleotide encodes an anti- CRISPR protein or an anti-CRISPR RNA. Examples of anti-CRISPR proteins include AcrE2, AcrE3, AcrE4, AcrFl, AcrF2, AcrF3, AcrF4, AcrF5, AcrF6, AcrF7, AcrF8, AcrF9, AcrFlO, AcrIIAl, AcrIIA2, AcrIIA3, AcrIIA4, AcrIICl, AcrIIC2, AcrIIC3 (Zhu et al., 2018).

[0144] In some embodiments, the neutralizing element encodes an anti- CRISPR protein selected from a type II-A anti-CRISPR protein A4 (AcrIIA4, SEQ ID NO:33) (Rauch et al., 2017; Shin et al., 2017; Taxiarchi et al., 2021), and a type V-A anti-CRISPR protein (AcrVA) (linek et al., 2012; Watters et al., 2018; H. Zhang et al., 2019), or variants thereof.

[0145] In some embodiments, the first polynucleotide or the third polynucleotide encode a CRISPR-based population suppression effector that expresses Cas9 or a variant thereof, including variants of Staphylococcus aureus Cas9 (SaCas9) and Streptococcus pyogenes Cas9 (SpCas9) (SEQ ID NO:31).

[0146] In some embodiments, the Cas9 variants are selected from a high- fidelity Cas9 variant such as eSpCas9(l.l), Cas9-HF1, HypaCas9, Cas9_R63A / Q768A, evoCas9, HiFi Cas9, Sniper-Cas9, and Sniper2L, a Cas9 PAM variant, a dCas9-FokI variant, a Cas9 nickase (Cas9n) (SEQ ID NO: 32), a dCas9 or Cas9n-based base editor, a Cas9n-based prime editor, a dCas9-based cleavage free editor, and a dCas9-based transposase or integrase.

[0147] In some embodiments, the first polynucleotide or the third polynucleotide encode a Casl2a-based population suppression effector that comprises Casl2a (Zetsche et al., 2015) (SEQ ID NO:30) or a variant thereof (Bandyopadhyay et al., 2020; L. Zhang et al., 2023).

[0148] In some embodiments, the first polynucleotide encodes a Cas9-based population suppression effector, and the second polynucleotide encodes a type II- A anti-CRISPR protein A4 (AcrIIA4) (Rauch et al., 2017; Shin et al., 2017; Taxiarchi et al., 2021, (SEQ ID NO:33)).

[0149] In some embodiments, the first polynucleotide encodes a Cas 12-based population suppression effector, and the second polynucleotide encodes a type V-A anti-CRISPR protein (AcrVA) (Watters et al., 2018; H. Zhang et al., 2019).

[0150] In some embodiments, the first polynucleotide encodes a CRISPR- based RNA-guided transcriptional activator (CRISPRa) population suppression effector that causes lethality or sterility by overexpression or ectopic expression of one or more endogenous genes, and the second polynucleotide encodes one of a recoded copy of one or more endogenous genes, an anti-CRISPR protein, and an anti-CRISPR RNA.

[0151] In some embodiments, one or more of the first, the second and the third polynucleotides encode for one or more protein coding genes that are controlled by one or more gene expression systems that allow expression of the protein coding genes in the animal, selected from a promoter (Nolan & Hammond, 2022), an inducible promoter (e.g. SEQ ID NO:68, 69), a repressible promoter (e.g. SEQ ID NO:70), a binary expression system, or a combination thereof.

[0152] In some embodiments, the expression system is driven by a promoter selected from a germline-specific promoter (e.g., SEQ ID NOs: 1-6, SEQ ID NOs:8-16, among others) a soma-specific promoter, a germline and soma promoter (e.g. SEQ ID NOs:7, 17, 22), a cell type-specific promoter, a tissue-specific promoter, a stage-specific promoter (Nolan & Hammond, 2022).

[0153] Promoter and terminator elements can be isolated from endogenous genes, and can promote broad expression across multiple cells, tissues, stages, and / or sexes, or they can be cell-, tissue-, stage-, and / or sex-specific. Promoters and terminator elements are selected from endogenous genes whose native expression profile matches the desired expression profile of the transgene. For example, a promoter sequence of a germline- specific gene will usually be capable of promoting germline- specific expression of a transgene. Typically, a promoter sequence will comprise a sequence of 250bp to 3000bp upstream of the start codon of the endogenous gene, and a terminator sequence will comprise from 250bp to lOOObp downstream of the stop codon. Specifically, promoter sequences are usually designed to incorporate the entire 5’ untranslated region (UTR), and terminator sequences by incorporating the entire 3’ UTR. More specifically, some genes produce multiple mRNA transcripts (i.e., splice variants) with different expression profiles, and promoters can be selected on the basis of a specific transcript such that the promoter and UTR sequences are designed on the basis of recapitulating expression of that splice variant, predicated on its specific start and stop codons. In some cases, the promoter sequences may be substantially longer than 3000bp, but it may be possible to recapitulate a substantially similar or desirable expression profile by taking only a fragment of the endogenous promoter sequence (Papathanos et al., 2009). In some cases, promoter sequences can be further modified by introducing, for example, mutations in or between transcription factor binding sites, so as to modify expression (Simoni et al., 2020b). Promoter sequences are often combined with their respective terminator sequence, but they can also be used with alternative terminators.

[0154] Oftentimes, a promoter sequence can be selected based on finding a gene that is present in a different animal species, searching for an ortholog of said gene, and identifying the putative promoter region. Such methods are well-known in the field and have been implemented and described at length (Nolan & Hammond, 2022).

[0155] In some cases, synthetic promoters can be used, such as 3xP3 (Horn & Wimmer, 2000), or viral sequences (e.g., Opie2, SV30, plO, etc). In other cases, endogenous promoter sequences from one species can be used to promote expression in another (Kandul et al., 2019; Yadav et al., 2023b). In some cases, a promoter can have its expression further limited by the inclusion of splice factors that limit the creation of mature mRNAs encoding full length protein sequences to the desired subset of cells, tissues, stages, or a specific sex (e.g. DSX-F in fsRIDL systems).

[0156] Promoter sequences can be further modified by the inclusion of enhancer sequences that typically alter the total expression levels, increase or decrease stability of the transcript, or alter expression specificity (e.g., plO, hr5).

[0157] Some promoters comprise one or more operator sequences that are placed in front of a minimal promoter sequence so that the entire promoter can activate transcription in response to a transactivator. Examples of such promoters include known binary expression systems and, typically, operator sequences are assembled into arrays (e.g. 7-21 repeats), and are placed in front of a minimal promoter sequence (such as hsp70) that will only allow substantial expression of the mRNA in presence of a transactivator. Example operator promoters include UAS of Gal4 / UAS expression systems, QUAS of Q-systems, tetO of tet-on and tet-off systems, and others.

[0158] Examples of expression systems include but are not limited to: a Gal4 / UAS system, a QF / QUAS system, a LexA system, an auxin-inducible system. In some embodiments, the transactivator associated to the expression system is inducible (e.g. SEQ ID NO:68, 69) or repressible (e.g. SEQ ID NO: 70) by a drug, such as tetracycline or doxycycline.

[0159] In some embodiments, the second polynucleotide is placed under the control of a binary expression system (e.g. UAS, QUAS, tetO), and the first polynucleotide expresses the transactivator of said binary expression system (e.g. Gal4, QF2, tTA, rtTA) such that the second polynucleotide is expressed only in animals also carrying the first polynucleotide. This configuration prevents expression of the neutralizing element in individuals that do not carry the first population suppression effector. In preferred embodiments the second polynucleotide is on the X chromosome, or on a region of the genome that shows strong preferential segregation into the female progeny of males (such as the non male-determining m-locus). This configuration ensures that male progeny of released males inherit the first polynucleotide but not the second, thus activating the first population suppression effector. In yet further embodiments, the second polynucleotide is in close genetic linkage with the third polynucleotide that is a repressible female lethality effector. In further preferred embodiments, the first polynucleotide expresses the transactivator under the same transcriptional control as the population suppression effector.

[0160] In some embodiments, the neutralizing element is an RNAi system targeting one or more effector mRNAs. Examples include a shRNA targeting Cas9, Cas9n, Casl2a, tetR, Bt toxin, and others.

[0161] In some embodiments, the first polynucleotide encodes an expression system that enables the expression of the RNAi system or of the targeted genome-editing system in the germline, in the soma, or in both the germline and the soma of the animal.

[0162] In some embodiments, the second polynucleotide encodes an expression system that enables the expression of the second polynucleotide only in the germline, only in the soma, or in both the germline and the soma of the animal.

[0163] In some embodiments, the first polynucleotide encodes an RNAi system that is targeted to impair the function of an essential gene, and the second polynucleotide encodes a functional recoded copy of said essential gene in which one or more nucleotides have been changed so that the gene cannot be targeted by the RNAi system (Buchman et al., 2018; Chen et al., 2007).

[0164] In some embodiments, the first polynucleotide encodes a targeted genome-editing system that creates null mutations in one or more essential genes (Yan et al., 2023) and the second polynucleotide encodes functional recoded copies of said essential genes that thus restore functionality of said essential genes (Adolfi et al., 2020).

[0165] In some embodiments, the essential gene or genes targeted by the first polynucleotide are required for male fertility (Figures 1, 15, 16), female fertility (Figures 3, 6, 14, 21, 22), male and female fertility, male viability (Figures 2, 17, 18), female viability, male fertility and female viability, male and female viability, or female sex determination.

[0166] In some embodiments, the animal has an XY system for sex determination and the first polynucleotide encodes an endonuclease that targets repetitive sequences present on the X chromosome. In a further embodiment, the endonuclease that targets repetitive sequences present on the X chromosome is inserted onto the Y chromosome or an autosome. Methods to identify such sequences on the X chromosome and target these for population suppression have been described previously (Papathanos & Windbichler, 2018; Tsoumani et al., 2020).

[0167] In some embodiments, the first polynucleotide, or the third polynucleotide, are a drug-repressible female lethality effector (Figures 13- 20, Figure 26).

[0168] In some embodiments, the first polynucleotide, or the third polynucleotide, is selected from a drug-repressible late-acting female lethality effector that kills the animal at a late stage of development, a drug- repressible post-zygotic female lethality effector that kills the animal after fertilization, and a drug-repressible embryonic female lethality effector that kills the animal after fertilization (Figures 13-20, Figure 26).

[0169] In some embodiments, the third polynucleotide encodes a construct selected from a RIDL construct or an fsRIDL construct and is located on the X chromosome, or within or in close linkage to the m-locus (Figures 13-20).

[0170] In some embodiments, the first polynucleotide, or third polynucleotide, is a temperature-inducible female lethality effector.

[0171] In some embodiments, the first polynucleotide, or third polynucleotide, is a female lethality effector that is repressible by a druginducible second polynucleotide (Figures 6-12, 23-25) (e.g. SEQ ID NOs:66, 67, 70).

[0172] In some embodiments, the first polynucleotide, or third polynucleotide, encodes a CRISPR-based population suppression effector that causes the death or sterility of a female, and a drug-inducible second polynucleotide is selected from a recoded copy of the endogenous gene targeted by said CRISPR-based population suppression effector, an anti- CRISPR protein and an anti-CRISPR RNA.

[0173] In some embodiments, the first polynucleotide encodes a male sterility and female lethality effector located on an autosome.

[0174] In some embodiments, the first polynucleotide encodes a male sterility and female lethality effector located on an autosome, and the second polynucleotide encodes an inducible neutralizing element genetically linked to the first polynucleotide (Figures 24 and 25).

[0175] In some embodiments, the first polynucleotide encodes a male sterility and female lethality effector located on an autosome, and the second polynucleotide is on the X chromosome (Figure 5).

[0176] In some embodiments the first polynucleotide encodes a male sterility and female lethality effector located on an autosome, and the second polynucleotide encodes an inducible neutralizing element on the X chromosome (Figure 12).

[0177] In some embodiments, the first polynucleotide encodes a male sterility and female lethality effector located on an autosome, and the second polynucleotide encodes an inducible neutralizing element located on an autosome.

[0178] In some embodiments, the second polynucleotide encodes AcrIIA4 on the X chromosome and, in further embodiments, the AcrIIA4 neutralizing element is inducible, for example by using a tet-on system (J. Chen et al., 2021, SEQ ID NO:41).

[0179] In some embodiments, the animal has an XY sex determination system, or an XO sex determination system, and the neutralizing element is on the X chromosome (Blackmon et al., 2017; De La Filia et al., 2015).

[0180] In some embodiments, the animal has an XY sex determination system, or an XO sex determination system, and the first polynucleotide encodes a male sterility and female lethality effector located on an autosome, and the second polynucleotide encodes an inducible neutralizing element on the X chromosome.

[0181] In some embodiments, the animal has a homomorphic sexdetermining chromosome system whereby one or more master-switch sexdetermining genes are on a chromosome that is mostly homomorphic (Blackmon et al., 2017). In some embodiments, the animal has a male-determining homomorphic sex-determining chromosome system whereby one or more male-determining genes are on a homomorphic sex chromosome within a heteromorphic region called the M-locus, and the heterologous minimally recombining region of the homomorphic sex chromosome is called the m- locus.

[0182] In some embodiments, the animal has a male-determining homomorphic sex-determining chromosome system, and the first polynucleotide is on the M-locus, or in tight genetic linkage with the M- locus. In related embodiments, the second polynucleotide is on the m-locus, or in tight genetic linkage with the m-locus.

[0183] In some embodiments, the first polynucleotide encodes a population suppression homing gene drive located on an autosome (Figure 23).

[0184] In some embodiments, the first polynucleotide encodes a population suppression homing gene drive that disrupts the sequence of a gene on an autosome that is required for female fertility or female viability (Figure 23).

[0185] In some embodiments, the first polynucleotide encodes a suppression homing gene drive that disrupts the sequence of a gene that is essential for females selected from, and including orthologs of, doublesex (SEQ ID NOs: 71-84), transformer and transformer-2, and sex-lethal, amongst others.

[0186] In some embodiments, the first polynucleotide encodes a split population suppression homing gene drive that disrupts the sequence of a gene essential for females on an autosome.

[0187] In some embodiments, the first polynucleotide encodes the gRNA component (e.g. SEQ ID NOs:42, 43, 45-50, 54-59) of a Y-linked split population suppression homing gene drive that disrupts the sequence of a gene essential for females located on an autosome, and the Cas9 component of said Y-linked split suppression homing gene drive is located on the Y chromosome (Gamez et al., 2023).

[0188] In some embodiments, the first polynucleotide encodes a male sterility effector located on an autosome or on the Y chromosome, and the second polynucleotide is located on the X chromosome.

[0189] In some embodiments, the first polynucleotide encodes a female lethality effector that causes death of the female progeny of the carrier by creating null mutations in female essential haploinsufficient genes or in X- linked haploinsufficient genes (Figures 3, 6, 14, 21-23).

[0190] In some embodiments the second polynucleotide and the third polynucleotide are in close genetic linkage that produces coinheritance of at least 99%, and in preferred embodiments they are in close genetic linkage on the X chromosome (Figures 13-22, and 27).

[0191] In some embodiments the first polynucleotide and the third polynucleotide are on an autosome in close genetic linkage that produces coinheritance of at least 99%. In a preferred embodiment thereof the second polynucleotide is on the X chromosome. In preferred embodiments thereof the first polynucleotide encodes a first population suppression effector selected from a male sterility effector, a male and female lethality effector, and a male sterility and female lethality effector.

[0192] In some embodiments, in the germline, the first polynucleotide generates heritable null mutations in somatically required haploinsufficient genes causing sterility, lethality, sex conversion, or intersex phenotypes in female, male, or both female and male progeny of the carrier (Figures 1-27).

[0193] In some embodiments the first polynucleotide generates an effector protein, RNA, or combination thereof that is deposited into the progeny in sufficient dose to generate sterility, lethality, sex conversion, or intersex phenotypes. In preferred embodiments, the effector is deposited paternally into embryos via the sperm.

[0194] In some embodiments, the first polynucleotide encodes a male sterility effector located on an autosome or on the Y chromosome, and the second polynucleotide and third polynucleotide are on the X chromosome (Figures 15 and 16). In some embodiments, the first polynucleotide encodes a male sterility effector located on an autosome or on the Y chromosome, and the second and third polynucleotide are in close genetic linkage on the X chromosome (Figures 15 and 16).

[0195] In some embodiments, the first polynucleotide encodes a targeted genome-editing system that creates mutations in one or more male fertility genes required for reproduction but not mating, and the second polynucleotide is located on the X chromosome.

[0196] In some embodiments, the second polynucleotide encodes one of an anti-CRISPR protein, an anti-CRISPR RNA, an RNAi system, and a recoded copy of a gene inactivated by the first polynucleotide (Figures 1- 27).

[0197] In some embodiments, the second polynucleotide is inducible by use of an inducible promoter, such as a tet-on system or an inducible RNAi system (Figures 6-12) (SEQ ID NO:41).

[0198] In some embodiments, the first polynucleotide encodes a sex-ratio distorter that biases the production of male progeny, such as an X-shredder, and the second polynucleotide encodes an inducible neutralizing element on the X chromosome (Figure 9). In further embodiments, said X-shredder is Ppol and the inducible rescue is an RNAi targeting Ppol. In yet further embodiments, the X-shredder is a CRISPR-based X-shredder and the inducible neutralizing element is an anti-CRISPR RNA or an anti-CRISPR protein such as AcrIIA4 (SEQ ID NO:34).

[0199] In some embodiments, animals carrying the first polynucleotide are generated by secondarily modifying animals carrying the second polynucleotide. Thus, the second polynucleotide can mitigate sterility, lethality or negative fitness effects caused by the first polynucleotide that would otherwise preclude or make difficult the generation of animals carrying the first polynucleotide.

[0200] The method of the present invention therefore allows for the reduction of a population of pest and / or invasive animals or disease vectors through the release of modified males that lead to a reduced reproduction of the target pest population. The method of the present invention lends itself particularly well to reducing the reproduction of populations of invertebrates that are pests and / or invasive or disease vectors, including species that are or could be damaging to human health, animal health, agriculture, forestry or the environment.

[0201] Due to the ever-changing environments and therefore ever-growing and evolving lists of pest and / or invasive species or disease vectors, a non- exhaustive list of preferred arthropod species that are or could be harmful to human activities or environment is reported here for exemplary and illustrative purposes:

[0202] Asian citrus psyllid (Diaphorina citri}, tomato leaf miner (Tuta absoluta}, Japanese beetle (Papilla japonica}, white-fringed beetle (Graphognatus spp.), citrus blackfly (Aleurocanthus woglumi}, Oriental fruit fly (Dacus dorsalis , tropical fruit fly (Dacus cucurbitae, Dacus zonatus}, Mediterranean fruit fly (Ceratitis capitata}, Natal fruit fly (Ceratitis rasa}, olive fruit fly (Batrocera oleae}, Queensland fruit fly (Bactrocera tryoni}, cherry fruit fly (Rhagoletis cerasi}, Caribbean fruit fly (Anastrepha suspensa}, whitefly (Bemisia tabaci}, fruit flies (Drosophila spp.), imported fire ants (Solenopis richteri, Solenopis invicta}, Gypsy moth (Lymantria dispar}, codling moth (Cydiapomonella}, brown tail moth (Euproctis chrysorrhoea}, diamondback moth (Plutella xylostella}, meadow spittlebug (Philaenus spumaris}, yellow fever mosquito (Aedes aegypti}, Asian tiger mosquito (Aedes albopictus}, common house mosquito (Culex pipiens}, malaria mosquitoes (Anopheles gambiae, Anopheles stephensi}, fall armyworm (Spodoptera frugiperda}, New World screwworm (Cochliomyia hominivorax}, Old World screwworm (Chrysomya bezziana}, corn rootworm (Diabrotica virgifera}, Tsetse fly (Glossina spp), Boll weevil (Anthonomous grandis}, red palm weevil (Rhynchophorus ferruginous}, Damsel fly Enallagma hageni), Dragonfly (Libellula luctuosa). brown- marmorated stink bug (Halyomorpha halys). rice stem borer (Tryporyza incertulas , European com borer Ostrinia nubilalis , and all other species that share genus with the aformentioned.

[0203] Expanding on the list presented above, sexually-reproducing pest and / or invasive species or disease vectors of the following Classes are suitable for the invention: Insecta, Entognatha, Arachnida, Chilopoda, Diplopoda, Symphyla, Branchiopoda, Cephalocarida, Branchiura, Pentastomida, Malacostraca.

[0204] Expanding on the list presented above, sexually-reproducing pest and / or invasive species or disease vectors of the following arthropod Orders are suitable and preferred for the invention: Ixodida (ticks), Trombidiformes and Sarcoptiformes (mites), Aranae (spiders), Scorpiones (true scorpions), Chilopoda (centipedes), Blattodea (cockroaches), Psocodea (lice), Hemiptera and Heteroptera (true bugs), Diptera (flies, midges and mosquitoes), Siphonaptera (fleas), Lepidoptera (butterflies and moths), Mecoptera (scorpionflies), Coleoptera (beetles), Orthoptera (locusts, grasshoppers), Notoptera (icebugs).

[0205] The invention can also be applied to control sexually-reproducing nematode pest and / or invasive species or disease vectors, including the following genera: Heterodera, Hoplolaimus, Meloidogyne, Pratylenchus, Rotylenchulus, and Xiphinema.

[0206] The present invention advantageously allows to flexibly adjust the persistence of the reduction of the reproduction of the animal population, depending on the combination of first, second, and, in some embodiments, third polynucleotides present in the released males.

[0207] Specifically, a reduction in reproduction lasting only one animal generation can be achieved, for example, by releasing males carrying only a first polynucleotide that causes death and / or sterility in the progeny or sterility of the carrier itself. The same effect can be achieved by releasing males also carrying a second polynucleotide that is inducible or repressible, but inactive in the released males.

[0208] Alternatively, a reduction of the reproduction of an animal population lasting precisely two animal generations can be achieved, for example, by releasing males carrying on the Y chromosome or homozygous on an autosome a first polynucleotide causing sterility of the wild-borne male progeny and, on the X-chromosome or homozygous on an autosome, a repressible third polynucleotide causing mortality or sterility of the wild- borne female progeny. The same effect can be achieved by releasing males also carrying a second polynucleotide that is inducible or repressible, but inactive in the released males. The same effect can also be achieved by releasing males carrying a first polynucleotide that is homozygous on an autosome that causes sterility of the carrier or lethality of its progeny and, on the X-chromosome, a second polynucleotide that fully mitigates or remedies the effect of the first polynucleotide and, on the X-chromosome or homozygous on an autosome, a repressible third polynucleotide causing mortality or sterility of the wild-borne female progeny. In this way, the male progeny of released males will not carry the second polynucleotide and thus, they will be effectively sterile, and the female progeny of released males will be killed by the third polynucleotide. Finally, a reduction of the reproduction lasting multiple animal generations can be achieved by, for example, releasing males carrying on the Y-chromosome or on an autosome a first polynucleotide causing sterility or death of the female progeny, and such reduction of the reproduction could be further augmented with a homing endonuclease, increasing its persistence over time.

[0209] Another important advantage of the invention is that it allows the mass-rearing of animals carrying one or more potential population suppression effectors on the first polynucleotide through the action of a second polynucleotide, which neutralises the population reduction effect of the first polynucleotide. Specifically, males can be easily reared such that they carry both the first and second polynucleotides and they can be mated to females also carrying the second polynucleotide such that the effect of the first polynucleotide is mitigated or remedied. The first polynucleotide can then be activated at the point of release by removal of the second polynucleotide through mating, wherein the males carrying the first polynucleotide but not the second polynucleotide can be isolated. If inserted on the X chromosome, the second polynucleotide can be isolated from the entire male progeny of modified males if said modified males are mated to unmodified females. Furthermore, if the X chromosome additionally contains the third polynucleotide, this allows the simultaneous separation of male progeny that carry only the first polynucleotide from the female progeny that carry both the second and third polynucleotides. Thus, this method allows to simplify mass rearing, sex sorting, and the release of modified males carrying a polynucleotide encoding a population suppression effector.

[0210] Similarly, an important novelty of the current invention is that it allows the generation of animals carrying lethal or sterile traits caused by the first polynucleotide by secondarily modifying animals carrying the second polynucleotide, thus achieving a temporary neutralisation of the lethal or sterile effect. In this way, animals can be easily modified to carry effectors, even if these generate lethality or sterility in both sexes. This contrasts with previous efforts at RIDL and fsRIDL generation, where it took many years to generate a small number of strains due to the delicate balance between the tetracycline repressor and RIDL-induced lethality at the point of creation. Furthermore, this method allows for the first time to generate strains carrying effectors of lethality, sterility, or that are otherwise highly deleterious that are not naturally inducible, nor repressible.

[0211] A further advantage of the current invention is that it allows conditional sex sorting by use of the inducible or repressible second polynucleotide, when the first polynucleotide is designed to induce female- specific lethality. In these cases, the animals reared in the presence of an inducer or in the absence or a repressor, thereby activating the neutralizing element, are unaffected. Prior to release, the inducer is removed or the repressor applied such that the female progeny of modified males are inviable and male-only releases can be achieved. As the inducer or repressor can be a drug added to the diet, this method enables a switch from mass rearing to sex sorting by a simply swapping between diets containing or lacking the drug.

[0212] Finally, in some embodiments, the method of the invention allows for an efficient mass-sexing through the use of a repressible third polynucleotide, for example when a repressible third polynucleotide causes female-specific lethality, such that males carrying the first polynucleotide (that causes the reduction in reproduction), the second polynucleotide (that allows an easy mass rearing) and the third polynucleotide can be separated from the females simply through the removal of said repressor.

[0213] The disclosures in Italian Patent Application No. 102023000023055 from which this application claims priority are incorporated herein by reference.

[0214] Where technical features mentioned in any claim are followed by reference signs, those reference signs have been included for the sole purpose of increasing the intelligibility of the claims and accordingly, such reference signs do not have any limiting effect on the interpretation of each element identified by way of example by such reference signs.

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Claims

CLAIMS1. Method for reducing the reproduction of a non-human, sexually reproducing, animal population comprising the steps of:(i) providing a genetically modified male carrying:- a first polynucleotide on the Y chromosome or on an autosome, said first polynucleotide encoding a first population suppression effector, selected from one or more of: a male and female lethality effector that causes death of the progeny of the carrier, a male and female sterility effector that causes sterility of the progeny of the carrier or causes sterility of the carrier itself, a male lethality effector that causes death of the male progeny of the carrier, a female lethality effector that causes death of the female progeny of the carrier, a female sterility effector that causes sterility of the female progeny of the carrier, a male sterility effector that causes sterility of the male progeny of the carrier or causes sterility of the carrier itself, a female-to-male sex conversion effector that causes a female progeny of the carrier to develop as a male, a male sterility and female lethality effector that causes sterility of the male progeny of the carrier or of the male carrier itself and death of the female progeny of the carrier or of the female carrier itself, and a male-sex distorter that causes the male carriers to sire a progeny which is more than 70% male; and- a second polynucleotide on the X chromosome or on an autosome, encoding a neutralizing element configured to mitigate or remedy the effect of said first population suppression effector;(ii) rearing the genetically modified male of step (i) by allowing it to reproduce with a genetically modified female comprising the said second polynucleotide on the X chromosome or on an autosome obtaining an animal population wherein the males carry both the first and the second polynucleotide;(iii) removing fertile females from the population obtained in step (ii);(iiia) optionally, rearing males obtained in step (iii) with a populationof non-genetically modified females and isolating the male offspring carrying only the first polynucleotide;(iv) releasing the animals obtained in step (iii) or the male offspring obtained in step (iiia) into the environment where a reduction of the reproduction of the animal is sought, obtaining a reduction of the reproduction of the animal population.

2. Method according to claim 1, wherein the method comprises step (iiia).

3. Method according to claim 1 or 2 wherein:(3a) the neutralizing element encoded by the second polynucleotide is a neutralizing element inducible by an inducer;(3b) rearing in step (ii) is conducted in presence of said inducer activating the inducible neutralizing element.

4. Method according to claim 1 or 2 wherein:(4a) the neutralizing element encoded by the second polynucleotide is a neutralizing element repressible by a repressor;(4b) rearing in step (ii) is conducted in absence of said repressor, thus activating the repressible neutralizing element.(4c) the males obtained in step (iii) or step (iiia) are treated with the repressor, thus inactivating the repressible neutralizing element.

5. Method according to claim 3 or 4, wherein the first polynucleotide is on an autosome.

6. Method according to claim 5, wherein the first and second polynucleotide are genetically linked within the same genomic locus, or are within one centimorgan of each other.

7. Method according to claims 3 to 6 wherein:(7a) the first population suppression effector is selected from a female lethality effector, a female sterility effector, a female-to-male sex conversion effector, a male sex distorter, a male and female sterility effector, and a male sterility and female lethality effector;(7b) removing fertile females in step (iii) is obtained by removing the inducer of step (3b), or by adding the repressor of step (4b), thus activating the first population suppression effector.

8. Method according to any of the preceding claims wherein:(8a) the genetically modified female of step (ii) additionally carries on an X chromosome or on an autosome a third polynucleotide encoding a second population suppression effector that is a female population suppression effector that is repressible by a repressor, said female population suppression effector being selected from a female lethality effector that, in absence of said repressor, causes death of the female carrier, a female sterility effector that, in absence of said repressor, causes sterility of the female carrier, a female-to-male sex conversion effector that, in absence of said repressor, causes sex conversion of female carriers into males, a male and female sterility effector that, in absence of said repressor, causes sterility of the progeny of the carrier or causes sterility of the carrier itself, and a male sterility and female lethality effector that, in absence of said repressor, causes sterility of the male progeny of the carrier or of the male carrier itself and death of the female progeny of the carrier or of the female carrier itself;(8b) rearing in step (ii) is conducted in presence of the repressor inactivating the second population suppression effector.(8c) removing fertile females in step (iii) is obtained by removing the repressor of step (8b).

9. Method according to claim 8 wherein the second and third polynucleotides are genetically linked within the same genomic locus, or are within one centimorgan of each other.

10. Method according to claims 8 or 9 wherein the first population suppression effector is selected from a female sterility effector, a female lethality effector, a male-sex distorter, and a female-to-male sex conversion effector.

11. Method according to claim 8 or 9 wherein the first population suppression effector is selected from a male sterility effector, a male and female lethality effector, and a male sterility and female lethality effector.

12. Method according to any of the preceding claims, wherein the first polynucleotide encodes a construct located on an autosome and is capable of homing in the germline.

13. Method according of any of the preceding claims wherein one or both of the first and the second population suppression effectors are selected from a targeted genome-editing system that creates one or more DNA sequence mutations that disrupt the function of one or more endogenous essential genes or sex-specific splice variants thereof, and an RNA interference system (RNAi) that disrupts the function of one or more endogenous essential genes or sex-specific splice variants thereof.

14. Method according to any of the preceding claims, wherein one or both of the first and the second population suppression effectors are selected from:- a CRISPR system, preferably selected from a CRISPR-Cas9 system and a CRISPR-Casl2a system, and- an RNA interference (RNAi) system, preferably a shRNA.

15. Method according to any of the preceding claims, wherein the first population suppression effector is a CRISPR system and the neutralizing element is selected from an anti-CRISPR protein or RNA, an RNAi configured to inactivate the CRISPR system, and a recoded copy of a gene inactivated by the first population suppression effector.

16. Method according to claim 15, wherein the neutralizing element is AcrIIA4.

17. Method according to any of the preceding claims wherein:(17a) the first population suppression effector encoded by the first polynucleotide is repressible by a repressor.(17b) rearing in step (ii) is conducted in presence of said repressor,thus inactivating the repressible first population suppression effector.

18. Method according to any of the preceding claims wherein the nonhuman, sexually reproducing, animal is an invertebrate pest and / or invasive species or vector of disease.

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