Mitochondrial female-associated lethality and universal sequence-specific toxin for animal population control

Abstract

A novel genetic circuit called MitoFail (mitochondrial female-associated lethality) that supports an animal-universal female-specific repressible lethality. This genetic circuit is built around universally active restriction endonucleases shredding mitochondrial DNA molecules (mtDNA) only in females. The essential nature of mtDNA for the animal viability and yet its vulnerability to the huge diversity of mitochondria-imported restriction endonucleases support the universal adaptability of MitoFail to divergent animal species. Since MitoFail is active only in females, it is passed down and spread by competitive males and results in the suppression of a targeted population by reducing female reproductive output. MitoFail offers multiple advantages over current genetic methods of insect control and is a promising addition to the genetic toolbox for controlling diverse animal species.

Description

PATENT APPLICATIONFORMITOCHONDRIAL FEMALE- ASSOCIATED LETHALITY AND UNIVERSAL SEQUENCE-SPECIFIC TOXIN FOR ANIMAL POPULATION CONTROLCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 515,299 filed July 24, 2023. which application is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present invention relates to genetic engineering strategies for population control.BACKGROUND

[0003] Insect control traditionally relies on insecticides and larvicides. However, consistent application of insecticides has promoted selection of resistance to applied chemicals in targeted species, while decimating off-target insect pollinators1 4and poisoning the environment. The warming and wetting climate has already resulted in the range expansion of many insect species exacerbating the pest and vector burden. These developments highlighted interest in traditional and novel biological approaches for insect control.

[0004] Sterile Insects Technique (SIT) is a species-specific and environmentally friendly approach for insect control involving massive releases of irradiated males that reduce wild insect numbers through sterile matings. SIT has been applied successfully to control a few insect species and can be extended to many insect species in which females mate only once in their lifetime5’6. However, it is an expensive and hard-to-scale approach that requires massive repeated releases to compensate for low competitiveness of irradiated males.

[0005] Alternative genetic approaches were recently developed to address shortcomings of a traditional SIT. Many genetic methods of insect control are built around the tetracycline-repressible (Tet-off) expression system7. At the core of the Tet-off system is the tetracycline-controlled transactivator (tTA) that combines the tetracycline-repressible DNA- binding domain (tetR) with the transcriptional trans-activating domain (VP 16). In the absenceof tetracycline, tetR binds the TetO sequence specifically and, to a smaller degree, other DNA sequences unspecifically, and VP 16 activates transcription of nearby genes. In the presence of tetracycline, tetR does not bind DNA sequences and VP 16 does not activate transcription. To induce repressible insect lethality, the tTA was used to trans-activate an apoptosis effector gene8-9or auto-activate itself10-13and cause rampant transcriptional activation.

[0006] To limit the leakiness of transcriptional activation and fitness costs in males, a female-specific intron was inserted into the coding sequence (CDS) of the tTA protein11’12, but not into the CDSs of the used effectors8-9. Nevertheless, the precise titration between good repression and complete activation of the current Tet-off systems and reliance on species- specific components have complicated the adaptation of these circuits to other insect species.SUMMARY OF THE INVENTION

[0007] In one aspect, the disclosure provides a genetic circuit that supports a repressible female-specific lethality across diverse animal species, in particular insect species, referred to as Mitochondrial Female-associated lethality (MitoFail). Mitochondria and mitochondrial DNA (mtDNA) genomes are essential for cellular respiration and conserved across diverse animal species. MitoFaiFs universal action is achieved by targeting restriction endonucleases into mitochondria and cutting mtDNA genomes during an entire insect development resulting in the lethality. To limit the lethality and the circuit-related fitness costs to the female sex, the female-specific intron is inserted into coding sequences of both trans-activator (aka. tetR) and effector (aka. restriction endonucleases). To achieve cross-species portability, MitoFail contains protein coding sequences, promoters, introns, and 3’UTR sequences known to be active cross diverse insect species. The DNA sequence specificity of diverse restriction endonucleases and the universal requirement of mtDNA quality control for animal viability support the precision, versatility, robustness, and engineering flexibility of MitoFail that are unattainable by current approaches for animal control. In summary, mitochondria-targeted restriction endonucleases are promising biological ‘toxins’ that are active in animals and can be selected to be highly species-specific or species-general.

[0008] Now, therefore, in one aspect the instant disclosure provides a method of establishing repressible female-specific lethality for an animal based on sex-specific gene expression of a selectable and universal toxin, comprising: introducing into the animal one or more exogenous nucleic acids encoding a mitochondria-targeted endonuclease under thecontrol of an inducible promoter system, wherein the mitochondria-targeted endonuclease, a portion of the inducible promoter system, or both comprise a sex-specific splicing module or sex-specific promoter element. In embodiments, the sex-specific splicing module or sexspecific promoter element prevents expression of the mitochondria-targeted endonuclease in male animals. In embodiments, the sex-specific splicing module is a female-specific intron. In embodiments, the female-specific intron comprises a stop codon which causes translation termination of the gene comprising the female-specific intron in male animals. In embodiments, the sex-specific splicing module is present in both a polynucleotide encoding the mitochondria-targeted endonuclease and a polynucleotide encoding the portion of the inducible promoter system. In embodiments, the inducible promoter system comprises promoter region operably coupled to the nucleic acid encoding the mitochondria-targeted endonuclease and a promoter element capable of binding to the promoter region and activate transcription of the mitochondria-targeted endonuclease. In embodiments, the inducible promoter system is a Tet-off expression system. In embodiments, the one or more exogenous nucleic acids further comprise a reporter gene. In embodiments, the method further comprises identifying sex-specific gene expression of the reporter gene to identify the animal as having the one or more exogenous nucleic acids. In embodiments, the promoter element is capable of being inhibited by an agent. In embodiments, the method further comprises supplying the agent to the animal to suppress the female-specific lethality. In embodiments, the animal is an insect. In embodiments, the animal is of the genus Drosophila. Also provided herein are populations of animals established according to the methods described herein.

[0009] In another aspect herein is a population of animals comprising one or more exogenous nucleic acids which encode a mitochondria-targeted endonuclease under the control of an inducible promoter system, wherein the mitochondria-targeted endonuclease, a portion of the inducible promoter system, or both comprise a sex-specific splicing module or a sexspecific promoter element. In embodiments, the sex-specific splicing module or sex-specific promoter element prevents expression of the mitochondria-targeted endonuclease in male animals. In embodiments, the sex-specific splicing module is a female-specific intron. In embodiments, the female-specific intron comprises a stop codon which causes translation termination of the gene comprising the female-specific intron in male animals. In embodiments, the sex-specific splicing module is present in both the mitochondria-targeted endonuclease and the portion of the inducible promoter system. In embodiments, the inducible promoter system comprises an inducible promoter element operably coupled to the nucleic acidencoding the mitochondria-targeted endonuclease and a transcriptional activator capable of binding to the inducible promoter element and activate transcription of the mitochondria- targeted endonuclease. In embodiments, the inducible promoter system is a Tet-off expression system. In embodiments, the one or more exogenous nucleic acids further comprise a reporter gene. In embodiments, the transcriptional activator is capable of being inhibited by an agent.In embodiments, the animals are insects. In embodiments, the insects are of the genus Drosophila.

[0010] Also described herein is a method for suppressing a wild population of animals, comprising releasing a population of animals described herein into the wild population of animals; and allowing the population of animals to mate with the wild population of animals, thereby suppressing the wild population of animals by causing selective death of female offspring.

[0011] In another aspect herein is a nucleic acid system for establishing repressible female-specific lethality for an animal based on sex-specific gene expression of a selectable and universal toxin, comprising: a first polynucleotide encoding a mitochondria-targeted endonuclease under the control of an inducible promoter system, wherein the mitochondria- targeted endonuclease and / or a portion of the inducible promoter system comprise a sexspecific splicing module or sex-specific promoter element. In embodiments, the system further comprises a second polynucleotide encoding the portion of the inducible promoter system. In embodiments, the first and second polynucleotides are present on the same nucleic acid. In embodiments, the inducible promoter system comprises an agent-responsive promoter element. In embodiments, the first polynucleotide comprises a promoter sequence operably coupled to the portion encoding the mitochondria-targeted endonuclease capable of being bound by a promoter element of the inducible promoter system. In embodiments, the system comprises the sex-specific splicing module. In embodiments, the sex-specific splicing module is a femalespecific intron. In embodiments, the female-specific intron comprises a stop codon. In embodiments, the female-specific intron is positioned in-frame with the sequence of the first polynucleotide. In embodiments, the female-specific intron is positioned in-frame with the portion of the inducible promoter system, optionally wherein the portion of the inducible promoter system is a repressor element capable of binding to a promoter sequence operably coupled to the first polynucleotide. In embodiments, the system further comprises a polynucleotide encoding a reporter element.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figures 1A-1B show: Mitochondrial Female-associated lethality (MitoFail) genetic circuit. MitoFail relies on a trans-activated mitochondria-targeted Restriction Endonuclease (mtRE) that is imported inside mitochondria and efficiently cut and linearize the circular mitochondrial DNA (mtDNA) molecules resulting in the organismic lethality (A). A conventional Tet-off expression system operates across diverse animal species and is used to transactivate the expression of mtRE. In the absence of tetracycline (Tet), the tTA protein containing two domains, tetR and VP 16, binds to tetO sequences and activates the transcription of mtRE. In the presence of Tet, Tet attaches to tetR blocking its binding to tetO and repressing the transcription activation, hence the system is called as Tet-off. To limit lethality and fitness costs associated with any potential leaky expression to females only, the female-specific intron is inserted inside coding sequences of tTA and mtRE. This intron is spliced out entirely in females (y), while in males ( ) an exon containing a stop-codon is retained causing a translation termination and absence of both tTA and mtRE proteins in MitoFail males. The MitoFail genetic construct was engineered to be functional across many species of fruit flies (B). The PacI endonuclease was used in MitoFail, since its recognition site is common in the AT-rich insect mtDNA. The mtDNA of Drosophila melanogaster and Drosophila suzukii harbors 21 and 10 PacI recognition sites, respectively. The DsRed2 fluorescent protein was used as a genetic marker of both MitoFail males and females. MitoFail was assembled inside a piggyBac (pBac) plasmid to facilitate transgenesis across diverse insect species. Finally, to insulate MitoFail from the position effect, g psy insulator sequences were used.DETAILED DESCRIPTION

[0013] The present disclosure relates, in part, to a method of establishing repressible female-specific lethality for a plurality of insects or other animals based on sex-specific gene expression of selectable and universal toxin.

[0014] In embodiments, the method comprises one or more of:(a) generating an exogenous nucleic acid molecule comprising a repressible femalespecific lethality system;(b) applying a mitochondria-targeted endonuclease as a sequence-specific toxin to induce organismic lethality via cutting mitochondrial DNA (mtDNA) or a plurality of insects and animal species;(c) delivering the exogenous nucleic acid molecule into an insect from the plurality of insects, wherein the exogenous nucleic acid molecule comprises a promoter region, a sequence-specific toxin, a sex-specific splicing module, a reporter gene, and a transcription terminator; (d) applying a sex-specific intron or promoter sequence to limit the expression of the sequence-specific toxin to one sex;(e) applying a Tet-off system to maintain transgenic strain on the tetracycline / doxycy cline-supplemented food;(f) inducing sex-specific lethality by di-repression of the sequence-specific toxin on regular food, not-supplemented with tetracycline / doxycycline; and / or(g) application of a system for control of the plurality of insects and beyond based on spread via unaffected males, thereby establishing a repressible female-specific lethality.

[0015] In embodiments, the exogenous nucleic acid molecule further comprises a piggy Bac inverted terminal repeat located at each end of the effector region.

[0016] In embodiments, the selectable toxin comprises a mitochondria-targeted PacI an OpIE-2 promoter.

[0017] In embodiments, the invention provides methods of suppressing an animal species based on expression of female-specific mitochondria-targeted endonuclease and spreading it via unaffected males. In embodiments, the method comprises: (a) generating an exogenous nucleic acid molecule comprising a sex-specific gene expression system; (b) delivering the exogenous nucleic acid molecule into an insect, wherein the exogenous nucleic acid molecule comprises a promoter region, a sex-specific splicing module, a reporter gene, and a transcription terminator; and (c) identifying sex-specific gene expression of the reporter gene, thereby identifying the insect based on the sex-specific gene expression.

[0018] In embodiments, the invention provides methods of inducing lethality a plurality of insects and animal species by cutting mtDNA by highly selectable restriction endonucleases, the method comprising:(a) generating an exogenous nucleic acid molecule comprising a selectable restriction endonuclease;(b) delivering the exogenous nucleic acid molecule into an insect, wherein the exogenous nucleic acid molecule comprises a promoter region, a sex-specific splicing module, a reporter gene, and a transcription terminator; an(c) identifying sex-specific gene expression of the reporter gene; thereby identify ing the insect based on the sex-specific gene expression.

[0019] Various further aspects and embodiments of the disclosure are provided by the following description. Before further describing various embodiments of the presently disclosed inventive concepts in more detail by way of exemplary description, examples, and results, it is to be understood that the presently disclosed inventive concepts are not limited in application to the details of methods and compositions as set forth in the following description.The presently disclosed inventive concepts are capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary, not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting unless otherwise indicated as so. Moreover, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the disclosure. However, it will be apparent to a person having ordinary skill in the art that the presently disclosed inventive concepts may be practiced without these specific details. In other instances, features which are well known to persons of ordinary skill in the art have not been described in detail to avoid unnecessary complication of the description. All of the compositions and methods of production and application and use thereof disclosed herein can be made and executed without undue experimentation in light of the present disclosure.REPRESSIBLE FEMALE-SPECIFIC LETHALITY SYSTEMS

[0020] In one aspect described herein is a system comprising one or more nucleic acids encoding components which provide for the establishment of repressible female-specific lethality of an animal (e.g., an insect). In embodiments, the system relies on sex-specific gene expression of a toxin which kills female animals of the species. In embodiments, the toxin targets mitochondrial DNA (mtDNA) of the animal. In embodiments, the toxin is a mitochondrial targeted endonuclease. In embodiments, the mitochondrial targeted endonuclease specifically targets regions of the mtDNA of the animal, thereby digesting the mtDNA and resulting in death of the animal. In embodiments, all female animals which expressthe toxin are non-viable. In embodiments, the system described herein is repressible, thus allowing for the toxin to be expressed only at desired time points or at desired situation. In embodiments, the system described herein provides for repressible expression of the toxin. In embodiments, the toxin is repressible by providing a repressing agent to the animals (e.g., an agent which prevents expression of the toxin). In such cases, the agent is one which can be readily provided to the animals and be readily removed from the environment of the animals, such as in a food supply (e.g., tetracycline as within a Tet-on or Tet-off expression system).

[0021] In embodiments, the system for establishing repressible female specific lethality7of an animal as described herein comprises the following components encoded on the one or more nucleic acids: 1) a toxin sufficient to kill the animals upon expression of the toxin (e.g., the mitochondrial targeted endonuclease); and 2) an inducible promoter system configured to allow for the toxin to be expressed only in a desired sex of the animal and offspring thereof (e.g., only kills female animals). In embodiments, at least one of the toxin and / or at least a portion of the inducible promoter system (e.g., at least one protein necessary for the function of the inducible promoter system) are placed under operative control of at least one sex-specific element (e.g., a sex-specific promoter, a sex-specific repressor, a sex-specific splicing module, etc.) which results in the toxin being expressed only in one sex of the animal (e.g., only- expressed in female animals). In embodiments, the system results in only or substantially only one sex of the animal being killed by the toxin.

[0022] In embodiments, the system for stablishing repressible female-specific lethality for an animal based on sex-specific gene expression of a selectable and universal toxin, comprises: a mitochondria-targeted endonuclease under the control of an inducible promoter system, wherein the mitochondria-targeted endonuclease, a portion of the inducible promoter system, or both comprise a sex-specific splicing module or sex-specific promoter element.

[0023] In embodiments, at least one of the toxin (e.g.. the mitochondrial targeted endonuclease) and / or a portion of the inducible promoter system (e.g., an enhancer or a repressor which interacts with a promoter region operably coupled to the gene expressing the toxin) is configured to be sex-specifically expressed (e.g., expressed only in the desired sex of organisms to be killed (e.g.. female insects)). Such sex-specific expression can be accomplished in a variety of methods, and each component (in cases where the toxin and the portion of the inducible promoter system are both configured to be sex-specifically7expressed) can be controlled by the same mechanism or a different mechanism (e.g., one component, suchas the toxin, can be controlled by a sex-specific splicing module and the other component, such as the portion of the inducible promoter system, can be controlled by a sex-specific promoter).

[0024] In embodiments, at least one of the toxin and / or a portion of the inducible promoter system is configured to be sex-specifically expressed in female animals. In embodiments, at least one of the toxin and / or the portion of the inducible promoter system is expressed under the control of a sex-specific promotor (e.g., a promoter required to express the toxin and / or the portion of the inducible promoter system is only expressed in female animals), a sex-specific repressor (e.g., a repressor which prevents expression of the toxin is expressed only in male animals), or the toxin and / or the portion of the inducible promoter system comprises a sex-specific splicing module in the gene encoding the toxin or the portion of the inducible promoter system (e.g., an intron which is only spliced out in a single sex). In embodiments, the sex-specific splicing module, the sex-specific promoter element, and / or the sex-specific repressor prevents expression of the toxin (e.g., the mitochondrial targeted endonuclease) in male animals.

[0025] In embodiments, both the toxin and the portion of the inducible promoter system are configured to be sex-specifically expressed in female animals. In embodiments, both the toxin and a portion of the inducible promoter system comprise a sex-specific splicing module. In embodiments, both the toxin and the portion of the inducible promoter system comprise a female-specific intron (i.e., an intron which is selectively spliced out only in female animals). In embodiments, the sex-specific splicing module is present in both a polynucleotide encoding the toxin (e.g.. the mitochondria-targeted endonuclease) and a polynucleotide encoding the portion of the inducible promoter system.

[0026] In embodiments, the portion of the inducible promoter system configured to be sex-specifically expressed in the animal is a promoter element (e.g., a promotor polypeptide) which facilitates the expression of the toxin, such as by binding to a promoter region operably coupled to a polynucleotide encoding the toxin (e.g., a tetracycline-controlled transactivator (tTA), such as those in the Tet-off system).

[0027] In embodiments, the system for the establishment of repressible female-specific lethality of an animal comprises a sex-specific splicing module (e.g., in the gene encoding the toxin or a portion of the inducible promoter system). In embodiments, the sex-specific splicing module is one which results in a lack of expression of an encoded protein or expression of anon-functional version of the encoded protein in one sex of the animal. In embodiments, the sex-specific splicing module is a sex-specific intron. In embodiments, the sex-specific intron comprises a stop codon in the open reading frame of the gene in which the sex-specific intron is present, thereby resulting in a lack of expression of the relevant encoded protein (e.g., the toxin or the portion of the inducible promoter system) in a functional form. In embodiments, the female-specific intron comprises a stop codon which causes translation termination of the gene comprising the female-specific intron in male animals. In embodiments, the sex-specific intron can result in a lack of expression of the relevant encoded protein (e g., the toxin or the portion of the inducible promoter system) in a functional form by causing a transcript of the gene to omit a necessary portion of the encoded protein. In embodiments, the sex-specific intron can be positioned within the gene at any desired position which results in a lack of expression of afunctional form of the encoded protein. In embodiments, the sex-specific intron is positioned at an interior position of the portion of the gene encoding the relevant protein (i.e., positioned downstream of at least one exon necessary for expression of the functional protein and up stream of at least one other exon necessary for expression of the functional protein). In embodiments, the sex-specific intron is positioned at an upstream position of the portion of the gene encoding the relevant protein (i.e., upstream of any exon necessary for the expression of the functional protein). In embodiments, the sex-specific splicing module is a female-specific intron (i.e., an intron which is selectively spliced out only in female animals).

[0028] In embodiments, a sex-specific splicing module of the instant disclosure comprises a female-specific intron from derived a transformer gene of an insect species (e.g., a species of fly). In embodiments, the sex-specific splicing module is derived from a transformer gene of a Ceratitis or Drosophila species (e.g.. Drosophila melanogaster or Ceratitis capitata). Alternative sex-specific splicing modules compatible with the instant disclosure are known in the art and are described in, for example, U.S. Pat. No. 10,844,402.

[0029] In embodiments, the expression of the toxin (e.g., the mitochondrial targeted endonuclease) is under the control of an inducible promoter. Many inducible promoters compatible with the instant disclosure are known in the art and can include without limitation Tet-on and Tet-off systems. In certain preferred embodiments, the inducible promoter used with a system of the instant disclosure is the Tet-off inducible promoter system.

[0030] In embodiments, an inducible promoter system used with a system as described herein will include at least one promoter element (e.g., a promoter polypeptide) and / or at leastone repressor element (e.g., a repressor polypeptide) which is capable of binding to a promoter region polynucleotide positioned on a nucleic acid encoding the toxin, thereby altering expression of the toxin. For example, in embodiments wherein the inducible promoter system comprises a promoter element which enhances or facilitates expression of the toxin upon binding to the promoter region, expression of the toxin will be increased, such as would be the case with a Tet-off or Tet-on system (e.g., increased to detectable or measurable levels, or increased by a factor such as at least 2-fold, 5-fold, 10-fold, 50-fold. 100-fold. 500-fold, or 100-fold). In other embodiments wherein the inducible promoter system comprises a repressor element, binding of the repressor element to the promoter region would block or inhibit expression of the toxin in desired situations.

[0031] In embodiments, the inducible promoter system comprises an inducible promoter element which is responsive to an agent. In embodiments, the inducible promoter element is capable of facilitating expression of the toxin or repressing expression of the toxin in response to an agent. Preferably, the agent can be readily administered to the animal containing the system in order to promote expression of the toxin at a desired time. For example, the agent could be administered to the animal during a rearing phase in which a population of the animals is allowed to propagate for later release into the wild. In such a case, it can be desirable to allow all offspring of the population to remain viable in order to facilitate generation of a suitably large population of the animals prior to release.

[0032] In embodiments, the system comprises a promoter element which is capable of being inhibited in its expression promoting activity by an agent. In such a case, in the absence of the agent, the promoter element is able to facilitate the expression of the toxin (e.g., the mitochondrial targeted endonuclease) by binding to a promoter region operably coupled to the toxin gene. Upon administration of the relevant agent, the abil i ty of the promoter element is inhibited in its ability to facilitate expression of the toxin. Non-limiting examples of such promoter elements include tetracycline-controlled transactivator (tTA) proteins of the Tet-off system which are controlled by the presence of tetracycline (i.e., the presence of tetracycline causes the tTA to not facilitate expression of the relevant gene). In such cases herein, such a promoter element can be referred to as an “agent-responsive” promoter element. Alternatively, the system could comprise a repressor element which is capable of binding to a promoter region operably coupled to the toxin gene which inhibits expression of the toxin in the presence of the agent.

[0033] In embodiments, the system described herein utilizes the Tet-off inducible promoter system. The Tet-off inducible promoter system comprises a tetracycline operator (tetO) as the promoter region which is capable of being bound by a tetracycline-controlled transactivator (tTA). The tTA of the Tet-off system is capable of binding to the tetO promoter region in the absence of tetracycline, thereby facilitating expression of the gene which is operably linked to the promoter region (e.g., the toxin). In the presence of tetracycline, the tTA of the Tet-off system is inhibited from binding to the tetO promoter region and thus expression of the gene which is operably linked to the promoter region (e.g., the toxin) is thereby inhibited. In embodiments, the system for or establishing repressible female-specific 1 ethal i ty described herein comprises nucleic acids which encode the tTA of the Tet-off system and which encode the toxin under the operative control of the tetO operator.

[0034] In embodiments, the tTA of the Tet-off system comprises a Tet repressor DNA binding protein (e.g., as derived from tetracycline resistance operon of Escherichia coli transposon TnlO) and one or more transcription activating domains, such as the VP16 from Herpes simplex virus. In embodiments, the ability of tTA to bind to the tetO promoter region is modulated by the presence or absence of tetracycline. In embodiments, when tetracycline is present, the tTA does not bind to tetO, thereby preventing transcription of the relevant encoded gene. In the absence of tetracycline, the tTA is able to bind to tetO, thereby facilitation transcription of the relevant encoded gene (e.g.. the toxin).

[0035] In embodiments, the system described herein comprises a universal toxin under the control of the inducible promoter, preferably a mitochondrial targeted endonuclease (e.g., a mitochondrial targeted restriction endonuclease). In embodiments, the mitochondrial targeted endonuclease comprises a mitochondrial matrix localization tag sequence. Preferably, the mitochondrial matrix localization tag sequence is selected from the same or a similar species of animal in which the system is used. However, as is apparent to one of ordinary skill in the art. mitochondrial-import machinery is highly conserved across animal groups, and so any suitable localization tag sequence can be selected.

[0036] In embodiments, the mitochondrial targeted endonuclease comprises a restriction endonuclease. In embodiments, the restriction endonuclease is selected based on its specificity for sequence in the mtDNA of the animal which are highly present in the mtDNA of the animal. For example, for D. melanogasier, PacI can be favorably selected as there are21 recognition sites of PacI in the D. melanogaster mtDNA in wild type animals. Selection ofsuitable restriction endonucleases for other species of animals can be readily performed by those skilled in the art by comparing the known recognition sites of various restriction endonucleases (e.g., those described in databases such as those provided at the Restriction Enzyme Database (REBASE®) maintained by New England BioLab) with known or readily ascertained mtDNA sequences of the relevant animals.

[0037] In embodiments, the one or more nucleic acids of the system for establishing repressible female-specific lethality are incorporated into the genome of the relevant animal (e.g., the insect). In embodiments, the one or more nucleic acids which encode the components of the systems as described herein are incorporated into the genome of an animal as described herein. Such techniques for the preparation of genetically modified organisms are known in the art and any suitable method can be used in order to prepare stock animals which contain the desired nucleic acids. In embodiments, the system comprises a plurality of nucleic acids which are integrated at different portions of the genome. In embodiments, the system comprises a single nucleic acid which is integrated into the genome at a single site (e.g.. the polynucleotide encoding the open reading frame of the relevant portions of the inducible promoter system and the polynucleotide encoding the open reading frame of the toxin are integrated into the genome as a single nucleic acid, with the portions of the inducible promoter system and the toxin separated by a suitable spacer group, such as a gypsy insulator sequence). In embodiments where the system further comprises a reporter element, such elements are also preferably incorporated into the genome in a single nucleic acid (e.g., all components of the system are incorporated by a single vector to ensure all elements are co-present). Such nucleic acids can be introduced into the genome by a wide variety of methods and using a wide variety of vectors, such as a Piggy Bae (pBac) vector. In embodiments, an insulator sequence (e.g., gypsy insulator sequence) is used between the open readings frames of each of the elements of the system described herein.

[0038] In embodiments, the nucleic acids encoding the relevant portions of the systems provided herein comprise additional elements for optimization of performance of the system. For example, the individual portions of the system can each independently comprise such additional elements, including without limitation promoters, enhancers, selection markers, reporter elements, transcription terminators, etc. In embodiments, each element of the system described herein comprises its own promoter region. Non-limiting examples of promoter regions comprise those associated with Opie2, Hr5IEl, U6 promoter, Act5C promoter, etc. Inembodiments, one or more of the elements can also comprise a transcription terminator, such as a plO 3’UTR, an Hsp70 terminator of D. melanogaster, and others known in the art.

[0039] In embodiments, the system provided herein can further comprise one or more reporter elements (or reporter genes encoding said reporter elements). In embodiments, the reporter element is one which provides a clear phenotypic difference between animals harboring the system provided herein and those who do not. (e.g., a fluorescent protein such as green fluorescent protein (GFP), dsRed, dsRed2, etc.). In embodiments, the reporter element is used to identify an animal as comprising the nucleic acids of the systems described herein.

[0040] Exemplary genetic elements of the system described herein are shown in FIG. 1A and FIG. IB. Turning to FIG. 1A, the system shown therein shows a first nucleic acid comprising the gene encoding the inducible promoter element tTA, which comprises a tetR domain and a VP 16 domain. Placed within the interior of the gene encoding the inducible promoter element tTA is a female-specific intron which contains a stop codon in the open reading frame, thus prevent expression of the tTA element in male animals. The nucleic acid shown contains a promoter region which is constitutively active, thus resulting in constant or near-constant expression in female animals where the intron is spliced out. Also depicted on the inducible promoter element is an Hsp70 3 UTR sequence as a transcription terminator which facilitates termination of the transcription of tTA, as well as degradation of the relevant mRNA. Also shown in FIG. 1A is the expressed tTA protein (circled tTA) and the promoter sequence of tetO on a second nucleic acid encoding the toxin (in this case, the mitochondrial targeted restriction endonuclease (mtRE)). The mtRR is flanked byHsp70 elements, including the Hsp70 3’ UTR. Like the gene encoding the promoter element tTA. the gene encoding the toxin also comprises a female-specific intron comprising an in-frame stop codon, thereby further preventing expression of the toxin in male animals.

[0041] FIG. IB shows a similar nucleic acid system to that of FIG. 1A, with the differences being that the system of FIG. IB contains a reporter element (DsRed2) and all the portions of the system are shown on a single vector (piggyBac vector). In FIG. IB, the reporter element is constitutively expressed under the control of strong promoter Hr5IEl with an SV40 enhancer element to ensure robust expression of the reporter, thereby facilitating sorting of animals which contain the desired nucleic acids. Following the reporter gene, a gypsy insulator sequence is shown to insulate expression of the cassette from position effects in the event of random genomic insertions. Following this, an identical gene encoding the toxin(mitochondrial targeted restriction endonuclease) as described in FIG. 1A is shown, followed by an identical inducible promoter element gene as also shown in FIG. 1 A, followed by a final gypsy insulator sequence.

[0042] In embodiments, the system for establishing repressible female-specific lethality as described herein is comprised in an animal (e.g., all of the required nucleic acids are incorporated into the genome of the animal). In embodiments, the animal is a pest animal. In embodiments, the animal is an insect. In embodiments, the insect is a pest insect. In embodiments, the insect is an aphid, ant, bee, beetle, cicada, cockroach, cricket, dragonfly, earwig, flea, fly, hornet, locust mantis, moth, mosquito, phasmid, silverfish, termite, tick, or wasp. In embodiments, the insect is a fly or a mosquito. In embodiments, the insect is a mosquito. In embodiments, the mosquito is of the genus Aedeomyia, Aedes, Anopheles, Armigeres, Ayurakitia, Borachinda, Coquillettidia, Culex, Culiseta, Deinocerites, Eretmapodites, Ficalbia, Galindomyia, Haemagogus, Heizmannia, Hodgesia, Isostomyia, Johnbelkinia. Kimia, Limatus, Lutzia, Malaya, Mansonia. Maorigoeldia, Mimomyia, Onirion, Opifex, Orthopodomyia. Psorophora, Runchomyia. Sabethes, Shannoniana. Topomyia, Toxorhynchites, Trichoprosopon, Tripteroides, Udaya, Uranotaenia, Verrallina, or Wyeomyia. In embodiments, the insect is a fly. In embodiments, the fly is of the genus Ceratitis or Drosophila. In embodiments, the fly is of the genus Ceratitis. In embodiments, the fly is Caratitis capitata. In embodiments, the fly is of the genus Drosophila. In embodiments, the fly is Drosophila melanogaster or Drosophila suzukii.

[0043] In embodiments, the instant disclosure also comprises populations of animals which comprise the system for establishing repressible female-specific lethality as described herein. In embodiments, the population comprises at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or more of the animals described herein. It is expressly contemplated that a “population” of animals as provided herein can comprise a single individual.

[0044] In embodiments, the animals comprising the system remain reproductively fit and viable in the absence of expression of toxin (e.g., the mitochondrial targeted endonuclease). In embodiments, male animals remain reproductively fit and viable in in the absence of an agent which suppresses expression of the toxin (e.g., in the absence of tetracycline for a system controlled by the Tet-off system as described herein). In embodiments, the animals are reproductively viable. In embodiments, the animals are able to reproduce (e.g., by mating) with wild type animals of the same type at a rate which is similar to a corresponding wild ty peanimal (e.g., one which does not contain the systems described herein). In embodiments, the animal comprising the system described herein is able to reproduce at least 25%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% as effectively as a corresponding wild type animal. In embodiments, the animal is able to compete with corresponding wild ty pe animals such that the animals comprising the system produce at least 25%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%. at least 75%, at least 80%, at least 85%. or at least 90% as many offspring as the corresponding wild type animals.

[0045] In embodiments, the animal comprising the system described herein has a survivability (e.g.. average lifespan) which is similar to that of a corresponding wild type animal. In embodiments, the animal comprising the system has a survivability (e.g., average lifespan) which is at least 25%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% that of a corresponding wild ty pe animal.

[0046] In embodiments, animals in which the toxin (e.g., the mitochondrial targeted endonuclease) becomes expressed are non-viable. In embodiments, the desired sex of animals become non-viable during an early stage of development (e.g., before maturity), such as at or before the pupal stage (when the animal is an insect).

[0047] Further provided herein in one aspect are nucleic acids useful in establishing the systems of sex-specific lethality’ (e.g., female-specific lethality) for an animal as described herein. In embodiments, such nucleic acids comprise a first polynucleotide encoding a mitochondria-targeted endonuclease under the control of an inducible promoter system, wherein the mitochondria-targeted endonuclease and / or a portion of the inducible promoter system comprise a sex-specific splicing module or sex-specific promoter element. In embodiments, the nucleic acids further comprise a second polynucleotide encoding the portion of the inducible promoter system. In embodiments, the first and second polynucleotides are present on the same nucleic acid, or the tw o polynucleotides can be on separate nucleic acids. In preferred embodiments, both polynucleotides are present on the same nucleic acid, thus ensuring all elements of the system can be integrated together. In embodiments, the inducible promoter system comprises an agent-responsive promoter element (e.g., of any of the inducible promoter systems described herein). In embodiments, the first polynucleotide comprises a promoter sequence operably coupled to the portion encoding the mitochondria-targetedendonuclease capable of being bound by a promoter element of the inducible promoter system. As will be apparent to one of ordinary skill in the art. the nucleic acids for establishing the systems described herein in an animal can include polynucleotides encoding any of the elements of the systems described herein (e.g., any of the reporter elements, promoters, enhancers, sex-splicing modules, etc.).

[0048] Further provided herein are methods of establishing the repressible sex-specific lethality (e.g., female specific lethality ) systems in an animal. In embodiments, such methods comprise introducing into a desired animal one or more exogenous nucleic acids encoding the elements of the systems described herein (e.g., one or more exogenous nucleic acids encoding a mitochondria-targeted endonuclease under the control of an inducible promoter system, wherein the mitochondria-targeted endonuclease, a portion of the inducible promoter system, or both comprise a sex-specific splicing module or sex-specific promoter element). In embodiments in which an agent-responsive control element (e.g., an agent responsive promoter element) are used, such methods can comprises supplying the agent to the animal in order to suppress the sex-specific lethality. For example, the agent could be supplemented into the food of the animal during growth and rearing stages to provide a stable population of animals which comprises the nucleic acids encoding the systems described herein.

[0049] In certain aspects, the systems described herein are useful in the suppression of a wild population of animals. For example, the systems described herein can be used to suppress, lower, or limit a wild animal population by releasing one or more animals comprising the nucleic acid systems herein which result in female-specific death of offspring upon breeding of the released animals with those in the wild population. Thus, one aspect of the instant disclosure provides method for suppressing a wild population of animals, comprising: releasing the population of animals comprising the system for establishing repressible female- specific lethality as described herein into the wild population of animals; and allowing the population of animals to mate with the wild population of animals, thereby suppressing the wi Id population of animals by causing selective death of female offspring. Consequently, the wild animal population is reduced in subsequent generations due to low levels of female animals in the next generation for subsequent breeding.

[0050] In embodiments, the method of suppressing the population of wild animals comprises iterative releases of the animals comprising the systems described herein. In embodiments, the method of suppressing the population of wild animals comprises iterativereleases of the animals comprising the systems described herein over a period of time, which can optionally be pre-set or determined in advance of initial release. In embodiments, further populations of the animals comprising the systems described herein are released over a period of at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 1.5 years, at least 2 years, at least 2.5 years, at least 3 years, at least 3.5 years, at least 4 years, at least 4.5 years, or at least 5 years. In embodiments, further populations are released or anticipated to be released for an indefinite period of time.

[0051] In embodiments, populations of the animals are released into the wild population according to a set schedule. In embodiments, populations of the animals are released, for example, daily, twice-weekly, three time a week, weekly, every 10 days, every two weeks, every three weeks, every four weeks, monthly, bi-monthly, every six weeks, every eight weeks, every two months, every three months, or at any appropriate interval.

[0052] In embodiments, the release of the animals is targeted to reduce the wild population of animals to an indicated threshold. In embodiments, the release of the transgenic animals is targeted to and effective to reduce the population of wild type animals by at least, for example, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% compared to the wild population before population suppression measures are taken.

[0053] In embodiments, the methods of population control provided herein can be utilized alongside other methods known in the art for controlling the population of animals. Such methods can include without limitation pesticide-based methods, Sterile Insect Techniques (SITs, such as precision guided SITs (pgSIT)), or other methods.

[0054] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0055] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein.

[0056] The practice of the present invention may employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ. Gait, ed.. 1984); Methods in Molecular Biology. Humana Press; Cell Biology; A Laboratory Notebook (J .E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed..1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993- 1998) J. Wiley and Sons; Methods in Enzy mology (Academic Press, Inc.); Handbook of Experimental Immunology (D .M. Weir and CC. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F .M. Ausubel et al , eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al, eds., 1994); Current Protocols in Immunology (J.E. Coligan et al, eds., 1991); Short Protocols in Molecular Biolog)’ (Wiley’ and Sons. 1999); Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary’ methods, devices, and materials are described herein. For the purposes of the present disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure. CERTAIN DEFINITIONS

[0057] As used herein, the terms "comprises." “comprising,” “includes,” “including,” “has.” “having,” “contains”, “containing,” “characterized by,” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a composition, and / or a method that “comprises” a list of elements (e.g., components, features, or steps) is not necessarily limited to only those elements (or components or steps), but may include other elements (or components or steps) not expressly listed or inherent to the composition and / or method.

[0058] Reference throughout this specification to “one embodiment,” “an embodiment.” “a particular embodiment,” “a related embodiment.” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of theforegoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0059] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0060] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a fusion protein, pharmaceutical composition, and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’. It is understood that aspects and embodiments of the invention described herein include “consisting” and / or “consisting essentially of’ aspects and embodiments.

[0061] When introducing elements of the present invention or the preferred embodiment(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0062] The term “and / or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression “A and / or B” is intended to mean either or both of A and B, i.e. A alone, B alone or A and B in combination. The expression “A, B and / or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination or A, B, and C in combination.

[0063] It should be understood that the description in range format is merely for convenience and brevity and should not be constmed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4. from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example. 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Values or ranges may be also be expressed herein as “about,” from “about” one particular value, and / or to “about” another particular value. When such values or ranges are expressed, other embodiments disclosed include the specific value recited, from the one particular value, and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments, “about” can be used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.

[0064] It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments, “about” can be used to mean, for example, a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In various embodiments, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0065] As used herein any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.

[0066] As used herein, “engineered” or “genetically modified” or “transformed” are used interchangeably, wherein a cell has been manipulated by means of molecular reprogramming of a genomic sequence (e.g. by restriction, insertion, deletion, or substitution). Said cells include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0067] As used herein, “inactivating mutations” are used to describe mutations, i.e., insertions, deletions, or substitutions, of genomic nucleic acids that result in lack of formation of a transcript or translated product compared to that of a wildtype, or naturally occurring, genomic nucleic acid sequence. An inactivating mutation functionally inactivates, or renders non-functional and / or inoperative, a naturally occurring nucleic acid sequence for expression.

[0068] As used herein, a “knockout” or “KO” is used to refer to genetic manipulation, wherein the manipulation results in a gene being made nugatory and / or the function of the gene is eliminated, either mostly or completely. A knockout may be achieved through various methods known in the art, for example, integration of a premature stop codon or insertions and / or deletions to the degree of rendering the gene inoperative.

[0069] As used herein, a “knockdown” or “KD” is used to refer to genetic manipulation, wherein the manipulation results in a gene's expression being reduced. A knockdown may be achieved through use of genetic modification resulting in the reduced transcription of a gene or by use of introducing an exogenous polypeptide encoding a short DNA or RNA oligonucleotide(s) that have a sequence complementary to either the gene or an mRNA transcript resulting in lack of abundance of functional gene transcript.

[0070] The terms “exogenous” and “heterologous” are used herein to refer to any molecule, including nucleic acids, protein or peptides, small molecular compounds, and the like that originate from outside the organism. In contrast, the term “endogenous” refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).

[0071] Genome editing tools may be used to engineer and / or manipulate genes in cells. In some embodiments, the immune cell of the disclosure may be engineered with either CRISPR. TALEN. or ZFN genome editing tools.

[0072] The target nucleic acid sequence can comprise 20 nucleotides. The target nucleic acid can comprise less than 20 nucleotides. The target nucleic acid can comprise more than 20 nucleotides. The target nucleic acid can comprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The target nucleic acid can comprise at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.

[0073] The mitochondria containing cells described herein can be modified using gene editing systems described herein to activate, inactivate, increase or reduce expression, or otherwise modify function of a target gene or a target gene product.

[0074] The term “nucleic acid7’ or “polynucleotide”, includes DNA and RNA such as genomic DNA, cDNA and mRNA, or combinations thereof. The nucleic acid may comprise, in addition to the sequence enabling the genetic modifications of the disclosure, further sequences such as those required for the transcription and / or translation of the nucleic acid enabling said genetic modifications. This may include a promoter, enhancer, transcription and / or translation initiation and / or termination sequences, selection markers, sequences protecting or directing the RNA and / or enabling the genetic modifications within the cell. The selection and combination of these sequences is within the knowledge of the person skilled in the art and may be selected in accordance with the cell the nucleic acid is intended for.

[0075] Polynucleotides enabling the genetic modifications of the disclosure may be delivered to cells as an isolated nucleic acid or in a vector. The isolated nucleic acid or the vector may be delivered in lipid- or lipid-based delivery’ system, such as a liposome.Alternatively, the vector may comprise viral proteins, such as when the vector is a viral vector. The term “vector” as used herein refers to a construction comprised of genetic material designed to direct transformation or transductions of a targeted cell. A vector contains multiple genetic elements positionally and sequentially oriented with other necessary elements such that the nucleic acid in a nucleic acid cassette can be transcribed and when necessary translated in the transfected cells. The term vector as used herein can refer to nucleic acid, e.g., DNA derived from a plasmid, cosmid, phagemid, bacteriophage, virus, retrovirus, adenovirus, adeno- associated virus, lentivirus, or other type of virus into which one or more fragments of nucleic acid may be inserted or cloned which encode for particular proteins. The term “plasmid” as used herein refers to a construction comprised of extrachromosomal genetic material, usually of a circular duplex of DNA which can replicate independently of chromosomal DNA. The plasmid does not necessarily replicate.

[0076] Any suitable vectors are envisaged as within the scope of the instant disclosure. The polynucleotides enabling the genetic modifications of the disclosure can be cloned into a number of types of vectors. For example, the polynucleotides enabling the genetic modifications of the disclosure may be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. Expression vectors may be provided to cells, such as immune cells, in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6.326,193).

[0077] The purpose of the vector is to provide a nucleic acid sequence in cells or tissue.Expression includes the efficient transcription of an inserted gene or nucleic acid sequence. Expression products may be proteins, polypeptides, or RNA. The nucleic acid sequence can be contained in a nucleic acid cassette. Expression of the nucleic acid can be continuous. constitutive, or regulated. The vector can also be used as a prokaryotic element for replication of plasmid in bacteria and selection for maintenance of plasmid in bacteria.

[0078] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0079] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0080] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0081] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0082] As used herein, and unless otherwise specified, the terms "prevent," "preventing" and "prevention" refer to the prevention of reproduction.

[0083] “Nucleic acid” or “nucleic acid molecule” refers to a multimeric compound comprising two or more covalently bonded nucleosides or nucleoside analogs having nitrogenous heterocyclic bases, or base analogs, where the nucleosides are linked together by phosphodiester bonds or other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and analogs thereof. A nucleic acid backbone can be made up of a variety of linkages, including one or more of sugar- phosphodiester linkages, peptide-nucleic acid bonds, phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of the nucleic acid can be ribose, deoxyribose, or similar compounds having known substitutions (e.g. 2'-methoxy substitutions and 2'-halide substitutions). Nitrogenous bases can be conventional bases (A, G, C, T, U) or analogs thereof (e.g., inosine, 5-methylisocytosine, isoguanine). A nucleic acid can comprise only conventional sugars, bases, and linkages as found in RNA and DNA. or can include conventional components and substitutions (e.g., conventional bases linked by a 2'- methoxy backbone, or a nucleic acid including a mixture of conventional bases and one or more base analogs). Nucleic acids can include “locked nucleic acids” (LNA), in which one or more nucleotide monomers have a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhances hybridization affinity toward complementary sequences in single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA). Nucleic acids can include modified bases to alter the function or behavior of thenucleic acid (e.g., addition of a 3'-terminal dideoxynucleotide to block additional nucleotides from being added to the nucleic acid). Synthetic methods for making nucleic acids in vitro are well known in the art although nucleic acids can be purified from natural sources using routine techniques. Nucleic acids can be single-stranded or double-stranded.

[0084] “Amplification” refers to any known procedure for obtaining multiple copies of a target nucleic acid or its complement, or fragments thereof. The multiple copies may be referred to as amplicons or amplification products. Amplification, in the context of fragments, refers to production of an amplified nucleic acid that contains less than the complete target nucleic acid or its complement, e.g., produced by using an amplification oligonucleotide that hybridizes to, and initiates polymerization from, an internal position of the target nucleic acid. Known amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), strand-displacement amplification (SDA), and transcription- mediated or transcription-associated amplification. Amplification is not limited to the strict duplication of the starting molecule. For example, the generation of multiple cDNA molecules from RNA in a sample using reverse transcription (RT)-PCR is a form of amplification. Furthermore, the generation of multiple RNA molecules from a single DNA molecule during the process of transcription is also a form of amplification. During amplification, the amplified products can be labeled using, for example, labeled pnmers or by incorporating labeled nucleotides.

[0085] “Amplicon” or “amplification product” refers to the nucleic acid molecule generated during an amplification procedure that is complementary or homologous to a target nucleic acid or a region thereof. Amplicons can be double stranded or single stranded and can include DNA, RNA or both. Methods for generating amplicons are known to those skilled in the art.

[0086] “Complementary” or “complement thereof’ means that a contiguous nucleic acid base sequence is capable of hybridizing to another base sequence by standard base pairing (hydrogen bonding) between a series of complementary bases. Complementary sequences may be completely complementary (i.e. no mismatches in the nucleic acid duplex) at each position in an oligomer sequence relative to its target sequence by using standard base pairing (e.g., G:C, A:T or A:U pairing) or sequences may contain one or more positions that are not complementary' by base pairing (e g., there exists at least one mismatch or unmatched base inthe nucleic acid duplex), but such sequences are sufficiently complementary because the entire oligomer sequence is capable of specifically hybridizing with its target sequence in appropriate hybridization conditions (i.e. partially complementary). Contiguous bases in an oligomer are typically at least 80%, preferably at least 90%, and more preferably completely complementary to the intended target sequence.

[0087] '‘Configured to” or “designed to” denotes an actual arrangement of a nucleic acid sequence configuration of a referenced oligonucleotide. For example, a primer that is configured to generate a specified amplicon from a target nucleic acid has a nucleic acid sequence that hybridizes to the target nucleic acid or a region thereof and can be used in an amplification reaction to generate the amplicon. Also as an example, an oligonucleotide that is configured to specifically hybridize to a target nucleic acid or a region thereof has a nucleic acid sequence that specifically hybridizes to the referenced sequence under stringent hybridization conditions.

[0088] "Downstream" means further along a nucleic acid sequence in the direction of sequence transcription or read out.

[0089] “Upstream” means further along a nucleic acid sequence in the direction opposite to the direction of sequence transcription or read out.

[0090] “Polymerase chain reaction"’ (PCR) generally refers to a process that uses multiple cycles of nucleic acid denaturation, annealing of primer pairs to opposite strands (forw ard and reverse), and primer extension to exponentially increase copy numbers of a target nucleic acid sequence. In a variation called RT-PCR, reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA. There are many permutations of PCR know n to those of ordinary skill in the art.

[0091] “Primer” refers to an enzymatically extendable oligonucleotide, generally with a defined sequence that is designed to hybridize in an antiparallel manner with a complementary, primer-specific portion of a target nucleic acid. A primer can initiate the polymerization of nucleotides in a template-dependent manner to yield a nucleic acid that is complementary' to the target nucleic acid wfien placed under suitable nucleic acid synthesis conditions (e.g. a primer annealed to a target can be extended in the presence of nucleotides and a DNA / RNA polymerase at a suitable temperature and pH). Suitable reaction conditionsand reagents are known to those of ordinary' skill in the art. A primer is ty pically single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is generally first treated to separate its strands before being used to prepare extension products. The primer generally is sufficiently long to prime the synthesis of extension products in the presence of the inducing agent (e.g. polymerase). Specific length and sequence will be dependent on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength. Preferably, the primer is about 5-100 nucleotides. Thus, a primer can be, e g., 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides in length. A primer does not need to have 100% complementarity with its template for primer elongation to occur; primers with less than 100% complementarity can be sufficient for hybridization and polymerase elongation to occur. A primer can be labeled if desired. The label used on a primer can be any suitable label, and can be detected by, for example, spectroscopic, photochemical, biochemical, immunochemical, chemical, or other detection means. A labeled primer therefore refers to an oligomer that hybridizes specifically to a target sequence in a nucleic acid, or in an amplified nucleic acid, under conditions that promote hybridization to allow selective detection of the target sequence.

[0092] A primer nucleic acid can be labeled, if desired, by incorporating a label detectable by, e.g.. spectroscopic, photochemical, biochemical, immunochemical, chemical, or other techniques. To illustrate, useful labels include radioisotopes, fluorescent dyes, electron- dense reagents, enzy mes (as commonly used in ELISAs), biotin, or haptens and proteins for which antisera or monoclonal antibodies are available. Many of these and other labels are described further herein and / or are otherwise known in the art. One of skill in the art will recognize that, in certain embodiments, primer nucleic acids can also be used as probe nucleic acids.

[0093] “Region” refers to a portion of a nucleic acid wherein said portion is smaller than the entire nucleic acid.

[0094] “RNA-dependent DNA polymerase” or “reverse transcriptase” (“RT”) refers to an enzyme that synthesizes a complementary DNA copy from an RNA template. All known reverse transcriptases also have the ability to make a complementary DNA copy from a DNA template; thus, they are both RNA- and DNA-dependent DNA polymerases. RTs may alsohave an RNAse H activity. A primer is required to initiate synthesis with both RNA and DNA templates.

[0095] “DNA-dependent DNA polymerase” is an enzyme that synthesizes a complementary DNA copy from a DNA template. Examples are DNA polymerase I from E. coli, bacteriophage T7 DNA polymerase, or DNA polymerases from bacteriophages T4, Phi- 29, M2, or T5. DNA-dependent DNA polymerases may be the naturally occurring enzymes isolated from bacteria or bacteriophages or expressed recombinantly, or may be modified or “evolved” forms which have been engineered to possess certain desirable characteristics, e.g., thermostability, or the ability to recognize or synthesize a DNA strand from various modified templates. All known DNA-dependent DNA polymerases require a complementary primer to initiate synthesis. It is known that under suitable conditions a DNA-dependent DNA polymerase may synthesize a complementary DNA copy from an RNA template. RNA- dependent DNA polymerases typically also have DNA-dependent DNA polymerase activity.

[0096] “Endonuclease” is an enzy me that cleaves the phosphodiester bond within a polynucleotide chain (namely DNA or RNA). Mitochondria-targeted Restriction Endonucleases (mtREs) are known in the art and used to effectively cut animal mt DNA17 iy.

[0097] A “sequence” of a nucleic acid refers to the order and identity of nucleotides in the nucleic acid. A sequence is typically read in the 5' to 3' direction. The terms “identical” or percent “identity” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, e.g., as measured using one of the sequence comparison algorithms available to persons of skill or by visual inspection. Exemplary algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST programs, which are described in, e.g., Altschul et al. (1990) “Basic local alignment search tool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification of protein coding regions by database similarity search” Nature Genet. 3:266-272, Madden et al. (1996) “Applications of network BLAST server” Meth. Enzymol. 266:131-141, Altschul et al. (1997) "’’Gapped BLAST and PSI- BLAST: a new generation of protein database search programs” Nucleic Acids Res. 25:3389- 3402, and Zhang et al. (1997) “PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation” Genome Res. 7:649-656, which areeach incorporated by reference. Many other optimal alignment algorithms are also known in the art and are optionally utilized to determine percent sequence identity.

[0098] “Hybridization,” “annealing,” “selectively bind,” or “selective binding” refers to the base-pairing interaction of one nucleic acid with another nucleic acid (typically an antiparallel nucleic acid) that results in formation of a duplex or other higher-ordered structure (i.e. a hybridization complex). The primary interaction between the antiparallel nucleic acid molecules is typically base specific, e.g., A / T and G / C. It is not a requirement that two nucleic acids have 100% complementarity' over their full length to achieve hybridization. Nucleic acids hybridize due to a variety of well characterized physio-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes part I chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” (Elsevier, New York), as well as in Ausubel (Ed.) Current Protocols in Molecular Biology. Volumes I. II, and III, 1997, which is incorporated by reference.

[0099] The term “attached” or “conjugated” refers to interactions and / or states in which material or compounds are connected or otherwise joined with one another. These interactions and / or states are typically produced by, e.g., covalent bonding, ionic bonding, chemisorption, physisorption, and combinations thereof.EXAMPLES

[0100] Methods and Results

[0101] The mtDNA genome is the small circular molecule, around 16,000 - 20,000 bases, found inside mitochondria. Each animal cell contains many mitochondria and hundreds of mtDNA molecules. While the mtDNA is mutating and evolving relatively fast at the DNA sequence level, all animal mtDNA genomes harbor identical genes essential for cellular respiration: 2 ribosomal RNAs, 22 transfer RNAs, and 13 protein-coding subunits of Electron Transport Chain (ETC)14’15. Multiple cellular pathways exist to ensure the quality control of multiple mtDNA molecules16>17, and the failures of these processes cause organismal diseases, aging, and death.

[0102] Mitochondria-targeted Restriction Endonucleases (mtREs) were used to effectively cut animal mtDNA17 19. The cut and linearized mtDNA molecules are unstable and quickly digested by exonucleases inside mitochondria. In fact, single-cutter mtREs that had a single recognition site in the Drosophila melanogaster wildtype mtDNA (mtDNAwt) was used to effectively cut all mtDNAwt: molecules and select for propagation of only the mtDNA molecules harboring preexistent and / or induced mutations at the endonuclease recognition sites and therefore resistant to cutting by the used mtREs 19. Double-cutter mtREs with two recognition sites in the mtDNAwt was used to induce the mtDNA molecules harboring large specific deletions (mtDNAA) that can co-occur in heteroplasmy with mtDNAwt molecules to study the mtDNA quality control pathways in post-mitotic cells1720. Finally, multiple-cutter mtREs were used to shred the mtDNAwt and obliterate a specific tissue or induce an organismic lethality (unpublished data). Therefore, by cutting animal mtDNA molecules, mtREs can act as a sequence-specific (aka. selectable or programmable) ‘toxin’ to induce an organismic lethality.

[0103] To assess the potential role of mtREs as a lethality inducing ‘toxin’, the expression of the mitochondria-targeted Pad (mtPacI) was compared with 21 recognition sites in the D. melanogaster mtDNAwt to that of the D. melanogaster head involuted defective (Hid), an activator of apoptosis. The overexpression of the Ras pathway insensitive Hid, HidAla521, was previously used to induce the lethality in insects and assemble genetic circuits for insect population suppression8’9’22’23. To limit the expression of functional proteins to females only, the Ceratitis capitate transformer female-species intron (CcTra ) was inserted into coding sequences of mtPacI and HidAla5. The CcTra intron splices completely in female mRNAs, while it leaves a stop containing male-specific exon in male mRNAs, which blocks translation of the proteins harboring CcTra in males24’23. To improve the expression level, the transcription of the CcTra harboring mtPacI and HidAla5, mt Pad CcTra andHidAla5CcTra (hereafter, mtPacI and HidAla5 ). respectively, was initiated from the Opie2 promoter and terminated with the plO 3'UTR. Both regulatory sequences, Opie226and plO27’28, originated from baculoviruses and are known to provide a strong constitutive expression in diverse insect species. Using these plasmids, two D. melanogaster transgenic strains were generated, each harboring Opie2-mtPad - l O or Opie2-HidAla5 -p l () integrated at the same genomic site using the phiC31 site-specific integration29, and compared the penetrance of induced female lethality.

[0104] It was found that mtPacI induced complete female lethality. No adult female harboring a single copy of Opie2-mtPacI -p lO has ever eclosed, while the adult females harboring Opie2-HidAla5$-plO survived and were fertile. As expected, both types of hemizygous males were not affected. The mating between Opie2-mtPacI$-pl 0 / + male and wt females produced only wt female progeny, and both wt and Opie2-mtPacI - l O / + males. To maintain the Opie2-mtPacI -p 10 strain, the hemizygous males were continuously out-crossed to wt females over ten generations, and no hemizygous females were ever observed. In the contrary, the hemizygous and homozygous Opie2-HidAla5 $-plO females were viable and fertile, though they had deformed wings and had some other abnormal features. The homozy gous Opie2-HidAla5 -pl O stock was established and maintained over eight generations. Overall these findings indicate that mtPacI is a more potent inducer of the female-specific lethality than HidAla5$ in D. melanogaster.

[0105] Next, to make the female-specific lethality repressible, mtPacI was placed under the Tet-off expression system7. The D. melanogaster vasa (Dmvas) or Opie2 promoter was used to initiate and the D. melanogaster Hsp70 3'UTR to terminate the transcription of tTA. To limit the fitness costs from a potential leaky7expression of tTA to females only, the CcTra intron was also inserted into the coding sequence of tTA. Under this circuit, the mRNAs of tTACcTra (hereafter, tTA$) were transcribed and degraded constitutively resulting in the stable level of the tTA protein only in females. In the absence of tetracycline. the tTA protein would bind specifically to TetO sequences and initiate the transcription of mtPacI using the basic D. melanogaster Hsp70 promoter, which would also be terminated with the D. melanogaster 3'UTR of Hsp70, resulting in continuous expressionthroughout the entire development. In the presence of tetracycline, the tTA protein would not bind to TetO and its mRNA would be degraded due to the Hsp70 3'UTR sequence. This repressible genetic circuit, called Mitochondrial Female-associated lethality or MitoFail (Fig.1A), was designed to be active only in females and not sacrifice male fitness, longevity , and competitiveness in the environment.

[0106] To ensure universal transgenicity across diverse insect species, MitoFail was assembled inside a PiggyBac (pBac) vector30-32. Both tTA and mtRE genes were surrounded by the gypsy insulator sequences33-34to insulate the expression of the MitoFail cassette from position effect35in random pBac-mediated genomic insertions. Finally, the DsRed2 fluorescent protein expressed under the Hr5IE136 promoter was added to trackinheritance of the MitoFail cassette. Multiple MitoFail strains were generated using sitespecific integration in D. melanogaster, and random pBac-mediated integration in both D. melanogaster and D. suzukii, a global agricultural pest, by rearing injected GO larvae on the tetracy cline / doxy cy cline-supplemented food (lOpg / ml).

[0107] As expected, the D. melanogaster and D. suzukii females harboring the MitoFail cassette required tetracycline to be supplemented to the food for their survival, otherwise the MitoFail females died during the development, by or at the pupal stage. A few versions of the D. melanogaster MitoFail strains were established and maintained as homozy gous stocks, in which females harbored two copies of the MitoFail genetic cassette. Notably, a few random insertion strains were established for both D. melanogaster and D. suzukii for which MitoFail / + females could not survive even on the tetracy cline / doxy cy cline-supplemented food, while the strains could be maintained by continuous backgrossing of hemizygous males to wt females. This suggests that the MitoFail genetic cassette indeed does not affect male fitness.

[0108] Finally, it w as validated that the tTA expression, even under the strong Hr5IEl promoter, did not induce the D. melanogaster female-specific lethality on its own, without the induced expression of mtPacI The generated D. melanogaster females harboring Hr5IEl- tTA$ on its own. without mtPacI . were viable and fertile. Therefore, a mitochondrial- targeted endonuclease, such as mtPacI ?, is absolutely required for the Mitofail’s functionality.

[0109] Discussion

[0110] The MitoFail genetic circuit has many beneficial features that make it customizable and adaptable for population control of diverse animals beyond insects.

[0111] The animal mtDNA contains identical genes that are essential to the Electron Transport Chain of Oxidative Phosphorylation and are highly conserved at the amino acid level. The mtDNA genomes are relatively short, i.e. Homo sapiens mtDNA is 16.6KB, Mus musculus mtDNA is 16.3KB, Aedes aegypti is 16.7KB Drosophila melanogaster mtDNA is 19.5KB, and known for the majority of animals or very easy and inexpensive to sequence. Identical mitochondrial matrix localization sequence tags are active across mammalian and insect species, since a highly conserved mitochondrial-import machinery operates in both diverse animal groups.

[0112] Animal mtDNA genomes are localized inside the mitochondrial matrix and are highly vulnerable to mtREs. Based on targeted mtDNA sequences, mtREs can be chosen to be general and cut mtDNAs across different species, for example mtPacI for the AT-rich mtDNA sequences of many insects, or species-specific and cut only a particular mtDNA genome of specific species harboring an unusual recognition site. Since mtREs are expected to work for nearly every animal and can be selected to be generalist or specialist, the mtRE is an ideal universal 'toxin’ that can be fine-tuned for each animal species.

[0113] The huge diversity' of restriction enzy me genes targeting unique recognition sites have been described and databased at the Restriction Enzyme Database (REBASE®) maintained by New England BioLab at rebase.neb.com37. REBASE facilitates the search and selection of a specific mtRE to fit a particular design of MitoFail.

[0114] The specificity or generality of a MitoFail genetic circuit can be further tuned by using regulatory DNA sequences such as promoters, introns, and 3'UTR that are known to be active across diverse taxa or species-specific.

[0115] The Tet-Off expression system has been developed to repress and transactivate, via di-repression. the expression of effector genes across diverse animal groups, from mammals to insects9’23’38’39, permitting the potential application of MitoFail across diverse animal species.

[0116] To limit MitoFail activity and any leakage of ‘toxicity’ induced by mtRE to females only, a female-specific intron was used. However, the female-specificity of MitoFail can be achieved also with female-specific promoters. The goal is preserve the fitness, longevity7, and competitiveness of MitoFail males to improve the efficacy7of MitoFail males to spread, compete with wt males, and mate with as many wt females as possible. In fact, the female-specific promoters that are active only in female reproductive tissues can also be used for expression of MitoFail. This MitoFail design will induce female sterility and cause suppression of a targeted animal species by reducing reproductive output of its females. This strategy' of reducing the female reproductive output with MitoFail may work well for rodent species.

[0117] Since mitochondria and mtDNA genomes are strictly7maternally inherited in the majority7of animal species, it is easy to reverse any unintended and adverse effects induced by a poor MitoFail system. The genetic cross with wt females will completely reseed healthy mtDNA genomes in the progeny.

[0118] In summary', the MitoFail genetic circuit offers many promising features facilitating its engineering and adaptation for control of insect and other animal species. Its reliance on cleavage of mtDNA molecules by endonucleases, such as restriction enzymes, to induce female-specific lethality or sterility opens doors to broad customization and nearly universal functionality. MitoFail will be a valuable addition to the genetic tools for control of pests, disease-vectors, and invasive animal species.

[0119] It will be understood from the foregoing description that various modifications and changes may be made in the various embodiments of the present disclosure without departing from their true spirit. The description provided herein is intended for purposes of illustration only and is not intended to be construed in a limiting sense. Thus, while the presently disclosed inventive concepts have been described herein in connection with certain embodiments so that aspects thereof may be more fully understood and appreciated, it is not intended that the presently disclosed inventive concepts be limited to these particular embodiments. On the contrary, it is intended that all alternatives, modifications and equivalents are included within the scope of the presently disclosed inventive concepts as defined herein. Thus the examples described above, which include particular embodiments, will serve to illustrate the practice of the presently disclosed inventive concepts, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of particular embodiments of the presently disclosed inventive concepts only and are presented in the cause of providing what is believed to be a useful and readily understood description of procedures as w ell as of the principles and conceptual aspects of the inventive concepts. Changes may be made in the construction and formulation of the various components and compositions described herein, the methods described herein or in the steps or the sequence of steps of the methods described herein without departing from the spirit and scope of the presently disclosed inventive concepts.REFERENCES

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Claims

AWhat is claimed is:1 . A method of establishing repressible female-specific lethality for an animal based on sex-specific gene expression of a selectable and universal toxin, comprising: introducing into the animal one or more exogenous nucleic acids encoding a mitochondria-targeted endonuclease under the control of an inducible promoter system, wherein the mitochondria-targeted endonuclease, a portion of the inducible promoter system, or both comprise a sex-specific splicing module or sex-specific promoter element.

2. The method of claim 1, wherein the sex-specific splicing module or sex-specific promoter element prevents expression of the mitochondria-targeted endonuclease in male animals.

3. The method of claim 1, wherein the sex-specific splicing module is a female-specific intron.

4. The method of claim 3, wherein the female-specific intron comprises a stop codon which causes translation termination of the gene comprising the female-specific intron in male animals.

5. The method of claim 1, wherein the sex-specific splicing module is present in both a polynucleotide encoding the mitochondria-targeted endonuclease and a polynucleotide encoding the portion of the inducible promoter system.

6. The method of claim 1, wherein the inducible promoter system comprises promoter region operably coupled to the nucleic acid encoding the mitochondria-targeted endonuclease and a promoter element capable of binding to the promoter region and activate transcription of the mitochondria-targeted endonuclease.

7. The method of claim 1 , wherein the inducible promoter system is a Tet-off expression system.

8. The method of claim 1, wherein the one or more exogenous nucleic acids further comprise a reporter gene.

9. The method of claim 8, further comprising identifying sex-specific gene expression of the reporter gene to identify the animal as having the one or more exogenous nucleic acids.

10. The method of claim 1, wherein the promoter element is capable of being inhibited by an agent.

11. The method of claim 10, comprising supplying the agent to the animal to suppress the female-specific lethality.

12. The method of claim 1, wherein the animal is an insect.

13. The method of claim 1, wherein the animal is of the genus Drosophila.

14. A population of animals established by the method of claim 1.

15. A population of animals comprising one or more exogenous nucleic acids which encode a mitochondria-targeted endonuclease under the control of an inducible promoter system, wherein the mitochondria-targeted endonuclease, a portion of the inducible promoter system, or both comprise a sex-specific splicing module or a sexspecific promoter element.

16. The population of claim 15, wherein the sex-specific splicing module or sex-specific promoter element prevents expression of the mitochondria-targeted endonuclease in male animals.

17. The population of claim 15, wherein the sex-specific splicing module is a femalespecific intron.

18. The population of claim 17, wherein the female-specific intron comprises a stop codon which causes translation termination of the gene comprising the femalespecific intron in male animals.

19. The population of claim 15, wherein the sex-specific splicing module is present in both the mitochondria-targeted endonuclease and the portion of the inducible promoter system.

20. The population of claim 15, wherein the inducible promoter system comprises an inducible promoter element operably coupled to the nucleic acid encoding the mitochondria-targeted endonuclease and a transcriptional activator capable of binding to the inducible promoter element and activate transcription of the mitochondria- targeted endonuclease.

21. The population of claim 15, wherein the inducible promoter system is a Tet-off expression system.

22. The population of claim 15, wherein the one or more exogenous nucleic acids further comprise a reporter gene.

23. The population of claim 15, wherein the transcriptional activator is capable of being inhibited by an agent.

24. The population of claim 15, wherein the animals are insects.

25. The population of claim 24, wherein the insects are of the genus Drosophila.

26. A method for suppressing a wild population of animals, comprising: releasing the population of animals of claim 15 into the wild population of animals; and allowing the population of animals to mate with the wild population of animals, thereby suppressing the wild population of animals by causing selective death of female offspring.

27. A nucleic acid system for establishing repressible female-specific lethality for an animal based on sex-specific gene expression of a selectable and universal toxin, comprising: a first polynucleotide encoding a mitochondria-targeted endonuclease under the control of an inducible promoter system, wherein the mitochondria-targeted endonuclease and / or a portion of the inducible promoter system comprise a sexspecific splicing module or sex-specific promoter element.

28. The nucleic acid system of claim 27, further comprising a second polynucleotide encoding the portion of the inducible promoter system.

29. The nucleic acid system of claim 28. wherein the first and second polynucleotides are present on the same nucleic acid.

30. The nucleic acid system of claim 27, wherein the inducible promoter system comprises an agent-responsive promoter element.

31. The nucleic acid system of claim 27. wherein the first polynucleotide comprises a promoter sequence operably coupled to the portion encoding the mitochondria- targeted endonuclease capable of being bound by a promoter element of the inducible promoter system.

32. The nucleic acid system of claim 27. comprising the sex-specific splicing module.

33. The nucleic acid system of claim 32, wherein the sex-specific splicing module is a female-specific intron.

34. The nucleic acid system of claim 33, wherein the female-specific intron comprises a stop codon.

35. The nucleic acid system of claim 33. wherein the female-specific intron is positioned in-frame with the sequence of the first polynucleotide.

36. The nucleic acid system of claim 33, wherein the female-specific intron is positioned in-frame with the portion of the inducible promoter system, optionally wherein the portion of the inducible promoter system is a repressor element capable of binding to a promoter sequence operably coupled to the first polynucleotide.

37. The nucleic acid system of claim 27, further comprising a polynucleotide encoding a reporter element.

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