WO2025166032A1 - Self-limiting, sex-specific transgenes and methods of use - Google Patents

Abstract

Provided herein are systems and methods for expressing a gene of interest in an organism. In some embodiments of the invention, the systems and methods provided herein are able to express the gene of interest in a sex-specific manner. In some embodiments of the invention, the systems and methods are provided for expression of a sex-specific lethal gene.

Description

SELF-LIMITING, SEX-SPECIFIC TRANSGENES AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/548,768, filed February 1, 2024, entitled “SELF-LIMITING, SEX-SPECIFIC TRANSGENES AND METHODS OF USE,” which is herein incorporated by reference in its entirety for all purposes.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (166372002440SEQLIST.xml; Size: 352,529 bytes; and Date of Creation: January 17, 2025) is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] Alternative splicing plays a key role in the regulation of gene expression in many developmental processes ranging from sex determination to apoptosis (Black, D. L. (2003) Annu. Rev. Biochem. 72, 291-336), and defects in alternative splicing have been linked to many human disorders (Caceres, J. F. & Kornblihtt, A. R. (2002) Trends Genet. 18, 186-193). In general, alternative splicing is regulated by proteins that associate with the pre-mRNA and function to either enhance or repress the ability of the spliceosome to recognize the splice site(s) flanking the regulated exon (Smith, C. W. & Valcarcel, J. (2000) Trends Biochem. Sci. 25, 381- 388).

[0004] Alternative splicing involves the removal of one or more introns and ligation of the flanking exons. This reaction is catalyzed by the spliceosome, a macromolecular machine composed of five RNAs, including small nuclear RNA and protein particles (snRNPs) which assemble with pre-mRNA to achieve RNA splicing, by removing introns from eukaryotic nuclear RNAs, thereby producing mRNA which is then translated to protein in ribosomes (Jurica, M. S. & Moore, M. J. (2003) Mol. Cell 12, 5-14; Smith, C. W. & Valcarcel, J. (2000) Trends Biochem. Sci. 25, 381-388). Alternative splicing generates multiple mRNAs from a single gene, thus increasing proteome diversity (Graveley, B. R. (2001) Trends Genet. 17, 100- 107).

[0005] Whether a particular alternative exon will be included or excluded from a mature RNA in each cell is thought to be determined by the relative concentration of a number of positive and negative splicing regulators and the interactions of these factors with the pre- mRNA and components of the spliceosome (Smith, C. W. & Valcarcel, J. (2000) Trends Biochem. Sci. 25, 381-388).

[0006] Malaria is a serious and sometimes fatal disease caused by a parasite that commonly infects certain types of mosquitoes. People who get malaria are typically very sick with high fevers, shaking chills, and flu-like illness. Five kinds of malaria parasites infect humans: Plasmodium falciparum, P. vivax, P. ovale, P. knowlesi, and P. malariae. P. falciparum and P. vivax pose the greatest threat. P. falciparum is the deadliest malaria parasite and the most prevalent on the African continent. P. vivax is the dominant malaria parasite in most countries outside of sub-Saharan Africa. Globally, the World Health Organization estimates that in 2020, 241 million clinical cases of malaria occurred, and 627,000 people died of malaria, most of them children in Africa. Because malaria causes so much illness and death, the disease is a great drain on many national economies. Since many countries with malaria are already among the poorer nations, the disease maintains a vicious cycle of disease and poverty.

[0007] Usually, people get malaria by being bitten by an infective female Anopheles mosquito. Only Anopheles mosquitoes can transmit malaria and they must have been infected through a previous blood meal taken from an infected person. When a mosquito bites an infected person, a small amount of blood is taken in which contains microscopic malaria parasites. About 1 week later, when the mosquito takes its next blood meal, these parasites mix with the mosquito’s saliva and are injected into the person being bitten.

[0008] Amongst the mosquito species that can transmit malaria, Anopheles stephensi and An. albimanus are two important vectors. An. stephensi is capable of transmitting both P. falciparum and P. vivax and An. albimanus can transmit P. vivax.

[0009] Vector control is a vital component of malaria control and elimination strategies as it is highly effective in preventing infection and reducing disease transmission. Two of the core interventions are insecticide-treated nets (ITNs) and indoor residual spraying (IRS).

[0010] Progress in global malaria control is threatened by emerging resistance to insecticides among Anopheles mosquitoes. As described in the latest World Malaria Report, other threats to ITNs include insufficient access, loss of nets due to the stresses of day-to-day life outpacing replacement, and changing behavior of mosquitoes, which appear to be biting early before people go to bed and resting outdoors, thereby evading exposure to insecticides. [0011] Additionally, although treatments are available for malaria, such as chloroquine phosphate and artemisinin-based combination therapies, there is an increasing risk of antimalarial drug resistance as a threat to global malaria control efforts.

[0012] Prevention of parasite transmission by vector mosquitoes has always played a major role in malaria control (Najera, Paras sitologia 43, 1-89 (2001); malERA Consultative Group on Vector Control, PLoS Med 8(l):el000401 (2011)). However, the current vector control methods have faced a slew of challenges including the heterogeneity and complexity of transmission dynamics and the difficulties in sustaining control practices (Luckhart et al., PLoS Negl Trop Dis 4, e566 (2010); Macias and James, Genetic Control of Malaria and Dengue, ed ZN Adelman (Elsevier Academic, San Diego), pp. 423-444 (2015)). Genetic approaches that result in altering vector populations in such a way as to eliminate their ability to transmit parasites to humans (population modification) can contribute to sustainable control and elimination by providing barriers to parasite and competent vector reintroduction, and allow resources to be directed to new sites while providing confidence that treated areas will remain malaria-free. With the potential for the spread of insecticide resistance, the development of transgenic vectors may provide an effective method to limit the transmission of the disease by reducing the density or vectoral capacity of the vector population.

[0013] Currently, in order to select only males, the pupae must be sorted by sex before eclosion, which could be labor-intensive and human error can result in sexing errors. It is also inefficient in medium to large-scale operational programs. Early and non-labor-intensive elimination of females could further enhance the cost saving benefit as potentially twice as many males could be produced from the same rearing environment as is currently possible.

[0014] There is a need in the art for a sex- specific self-limiting technology that effectively transmits the self-limiting technology to relevant mosquito populations while increasing the accuracy and efficiency of male/female separation.

[0015] We have developed and tested a self-limiting transgenic technology that confers a repressible phenotype whereby, in the absence of a tetracycline analogue, female mosquitoes carrying at least one copy of the transgene die at an early larval stage due to sex-specific expression and accumulation of an effector protein produced by a positive feedback loop. In another approach, we developed a system whereby in the absence of a tetracycline analogue, female mosquitoes carrying at least one copy of the transgene die due to expression of a female- specific lethal protein. In both approaches, male mosquitoes, which do not bite or transmit disease, are released to mate with wild females and therefore, the female progeny, which inherit and express the self-limiting gene, do not survive to adulthood due to the lack of tetracycline or its analogues in the environment. In generating mosquitoes for release, the larvae and pupae are grown in the absence of tetracycline or its analogues, which enables male mosquitoes, but not female mosquitoes, to survive to adulthood.

BRIEF SUMMARY OF THE INVENTION

[0016] The present invention provides a splice control module for differentially expressing a gene of interest in an organism. Also provided is a gene expression polynucleotide comprising a sex-specific lethal gene. Additionally, the present invention provides an expression vector plasmid for the splice control module or gene expression polynucleotide, a genetically engineered insect using the systems disclosed herein, methods of producing such a genetically engineered insect, methods of selectively rearing male genetically engineered insects, and methods of reducing a wild insect population.

[0017] In some embodiments, provided herein is a doublesex (dsx') splice control module polynucleotide comprising, from 5’ to 3’: i. a dsx exon 4 sequence; ii. a dsx intron 4 sequence; iii. a first portion of a dsx exon 5 sequence; iv. an effector protein coding sequence; v. a second portion of the dsx exon 5 sequence; vi. a dsx intron 5 sequence; and vii. a dsx exon 6 sequence.

[0018] In some embodiments, the dsx splice control module polynucleotide is derived from an Anopheles dsx. In some embodiments, the dsx splice control module polynucleotide is derived from an Anopheles stephensi (AsleDsx), Anopheles albimanus (AalhDsx). Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dims, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, or Anopheles gambiae dsx. In some embodiments, the dsx splice control module polynucleotide is derived from Anopheles stephensi dsx (AsleDsx). In some embodiments, the dsx splice control module polynucleotide is derived from Anopheles albimanus dsx AalbDsx).

[0019] In any of the embodiments disclosed herein, the first portion of the dsx exon 5 sequence can comprise a 5’ terminal fragment of dsx exon 5. In any of the embodiments disclosed herein, the second portion of the dsx exon 5 sequence can comprise a 3’ terminal fragment of dsx exon 5.

[0020] In any of the embodiments disclosed herein, the effector protein can be lethal, deleterious, or sterilizing to an insect. In any of the embodiments disclosed herein, expression of the effector protein can cause lethality in pre-adult females in the absence of tetracycline or an analogue thereof, with at least 90% effectiveness. In any of the embodiments disclosed herein, the effector protein can be selected from the group consisting of: a tTA or a tTAV gene variant, or a variant thereof, ReaperKR or a variant thereof, an apoptosis-inducing factor or a variant thereof, Hid or a variant thereof, and NipplDm or a variant thereof. In some embodiments, the effector protein is tTAV, tTAV2, or tTAV3. In some embodiments, the effector protein coding sequence comprises the sequence set forth in any of SEQ ID NOs: 18, 20, and 22. In some embodiments, the effector protein coding sequence comprises the sequence set forth in any of SEQ ID NOs: 26, and 30.

[0021] In any of the embodiments disclosed herein, the dsx exon 4 sequence can be modified compared to a wild-type dsx exon 4 sequence and/or the first portion of the dsx exon 5 sequence can be modified compared to a wild-type dsx exon 5 sequence to remove one or more start codons. In any of the embodiments disclosed herein, the dsx exon 4 sequence can be modified compared to a wild-type dsx exon 4 sequence and/or the first portion of the dsx exon 5 sequence can be modified compared to a wild-type dsx exon 5 sequence to remove one or more stop codons. In any of the embodiments disclosed herein, the modified dsx exon 4 sequence and/or first portion of the dsx exon 5 can comprise one or more modifications selected from the group consisting of a substitution, an insertion, and a deletion. In any of the embodiments disclosed herein, dsx splice control module polynucleotide may but does not need to comprise any open reading frame 5’ of the effector protein coding sequence.

[0022] In any of the embodiments disclosed herein, the splice control module polynucleotide can be spliced on a sex-specific basis when introduced into an insect to produce one or more female splice forms in female insects and one or more male splice forms in male insects. In some embodiments, the female splice forms express the effector protein. In some embodiments, the male splice form expresses the effector protein. In some embodiments, the male splice form does not express the effector gene in significant quantities. In some embodiments, the one or more female splice forms comprise, from 5’ to 3’: i. a dsx exon 4 sequence; ii. a first portion of a dsx exon 5 sequence; iii. an effector protein coding sequence; iv. a second portion of a dsx exon 5 sequence; and vi. a dsx exon 6 sequence. In some embodiments, the splice control module is capable of producing a plurality of different female splice forms in female insects, and wherein the length of the first portion of the dsx exon 5 sequence is variable between the plurality of different female splice forms.

[0023] In any of the embodiments disclosed herein, the splice control module can further comprise a 3’ UTR sequence. In some embodiments, the 3’ UTR sequence is derived from a PIO 3’ UTR or a SV40 3’ UTR. In some embodiments, the 3’ UTR sequence is derived from PIO 3’ UTR comprising the sequence set forth in SEQ ID NO: 33. In some embodiments, the 3’ UTR sequence is derived from SV40 3’ UTR comprising the sequence set forth in SEQ ID NO: 34.

[0024] In some embodiments, disclosed herein is a doublesex (dsx) splice control module polynucleotide operably linked to an effector protein coding sequence, comprising, from 5’ to 3’: i. a dsx exon 4 sequence; ii. a dsx intron 4 sequence; iii. a dsx exon 5 sequence; iv. a dsx intron 5 sequence; and v. a dsx exon 6 sequence, wherein the effector protein coding sequence is 3’ of the dsx splice control module polynucleotide.

[0025] In some embodiments, the dsx splice control module polynucleotide is derived from an Anopheles dsx.

[0026] In any of the embodiments disclosed herein, the effector protein can be tTAV, tTAV2, or tTAV3

[0027] In any of the embodiments disclosed herein, the splice control module polynucleotide can be spliced on a sex-specific basis when introduced into an insect to produce one or more female splice forms in female insects and one or more male splice forms in male insects.

[0028] In any of the embodiments disclosed herein, the effector protein coding sequence comprises the sequence set forth in any of SEQ ID NOs: 18, 20, and 22. [0029] In some embodiments, disclosed herein is a gene expression polynucleotide, comprising: a first module comprising from 5’ to 3’: i. a promoter operable in an insect; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR; and a second module comprising from 5’ to 3’: i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a female- specific lethal protein; and iv. a 3’ UTR.

[0030] In some embodiments, the female- specific lethal protein is lethal to female insects. In any of the embodiments disclosed herein, the female- specific lethal protein can be YOB or a variant thereof, or GUY 1 or a variant thereof. In any of the embodiments disclosed herein, the female- specific lethal protein can comprise the sequence set forth in any of SEQ ID NOs: 29 and 32. In any of the embodiments disclosed herein, the coding sequence of the female- specific lethal protein comprises the sequence set forth in any of SEQ ID NOs: 28 and 31.

[0031] In any of the embodiments disclosed herein, the promoter of the first module can be selected from the group consisting of bZIPl, GUY1 promoter, Vasa, Tre3G, Drosophila melanogaster minimal HSP70 promoter (DmHsp70), CMV minipromoter, OpIE2, and Act5c. In any of the embodiments disclosed herein, the first module can further comprise a 5’ UTR. In some embodiments, the 5’ UTR of the first module is derived from a GUY1 5’ UTR comprising the sequence set forth in SEQ ID NO: 66. In any of the embodiments disclosed herein, the promoter of the first module can comprise a sequence as found in any one of SEQ ID NOs: 36, 39, 40, and 65.

[0032] In any of the embodiments disclosed herein, the coding sequence of tTAV or a variant thereof can comprise the sequence set forth in any of SEQ ID NOs: 18, 20, and 22.

[0033] In any of the embodiments disclosed herein, the 3’ UTR of the first module can be derived from a K10 3’ UTR or a SV40 3’ UTR. In some embodiments, the 3’ UTR of the first module is derived from K10 3’ UTR comprising the sequence set forth in SEQ ID NO: 56. In some embodiments, the 3’ UTR of the first module is derived from SV40 3’ UTR comprising the sequence set forth in SEQ ID NO: 34.

[0034] In any of the embodiments disclosed herein, the promoter of the second module can comprise a sequence as found in SEQ ID NO: 36.

[0035] In any of the embodiments disclosed herein, the 5’ UTR of the second module can be derived from YOB 5’ UTR comprising the sequence set forth in SEQ ID NO: 67. In any of the embodiments disclosed herein, the 5’ UTR of the second module can be derived from GUY1 5’ UTR comprising the sequence set forth in SEQ ID NO: 66.

[0036] In any of the embodiments disclosed herein, the 3’ UTR of the second module can be derived from PIO 3’ UTR comprising the sequence set forth in SEQ ID NO: 33. In any of the embodiments disclosed herein, the 3’ UTR of the second module can be derived from YOB 3’ UTR comprising the sequence set forth in SEQ ID NO: 68. In any of the embodiments disclosed herein, the 3’ UTR of the second module can be derived from GUY1 3’ UTR comprising the sequence set forth in SEQ ID NO: 69.

[0037] In some embodiments, the gene expression polynucleotide disclosed herein comprises: a first module comprising from 5’ to 3’: i. a bZIPl promoter; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR selected from the group consisting of: SV40 and K10; and a second module comprising from 5’ to 3’: i. a TRE3G promoter; ii. a YOB 5’ UTR; iii. a YOB coding sequence; and iv. a 3’ UTR selected from the group consisting of: YOB and plO.

[0038] In some embodiments, the gene expression polynucleotide disclosed herein comprises: a first module comprising from 5’ to 3’: i. a promoter selected from the group consisting of: bZIPl, GUY1, Vasa, and TRE3G; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR selected from the group consisting of: SV40 and K10; and a second module comprising from 5’ to 3’ : i. a TRE3G promoter; ii. a GUY 1 5’ UTR; iii. a GUY 1 coding sequence; and iv. a 3’ UTR selected from the group consisting of: GUY1 and plO.

[0039] In any of the embodiments disclosed herein, the insect can be of the Order Diptera. In any of the embodiments disclosed herein, the insect can be a mosquito of a genus selected from the group consisting of Anopheles, Stegomyia, Aedes, and Culex. In some embodiments, the mosquito is a species selected from the group consisting of Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles arabiensis, Anopheles coluzzii, and Anopheles gambiae. In any of the embodiments disclosed herein, the insect can be a Calliphoridae species selected from the group consisting of Cochliomyia hominivorax, Chrysomya bezz.iana. and Lucilia cuprina. In any of the embodiments disclosed herein, the insect is a Diptera of a species selected from the group consisting of Ceratitis capitata, Anastrepha ludens, Bactrocera dorsalis, Bactrocera oleae, Bactrocera cucurbitae, Ceratitis rosa, Rhagoletis cerasi, Bactrocera tyroni, Bactrocera zonata, Anastrepha suspense, and Anastrepha obliqua.

[0040] In some embodiments, provided herein is a gene expression system comprising the dsx splice control module polynucleotide disclosed herein.

[0041] In some embodiments, the gene expression system disclosed herein further comprises a 5’ untranslated region (5’ UTR) operably linked 5’ of the dsx splice control module polynucleotide.

[0042] In some embodiments, the gene expression system disclosed herein further comprises a promoter operable in an insect. In some embodiments, the promoter is selected from the group consisting of Drosophila melanogaster minimal HSP70 promoter (DmHsp70), Tre3G, CMV minipromoter, OpIE2, Vasa, bZIPl, and Act5c.

[0043] In any of the embodiments disclosed herein, the gene expression system can comprise a UTR sequence as found in any one of SEQ ID NOs: 35-41.

[0044] In any of the embodiments disclosed herein, the promoter comprises a sequence set forth in any one of SEQ ID NOs: 42-44.

[0045] In any of the embodiments disclosed herein, the gene expression system disclosed herein can further comprise a tetracycline responsive operator.

[0046] In some embodiments, provided herein is an expression vector plasmid comprising the gene expression system disclosed herein or the gene expression polynucleotide disclosed herein.

[0047] In some embodiments, the expression vector plasmid disclosed herein further comprises a polynucleotide encoding a color marker protein. In some embodiments, the color marker protein is a fluorescent marker protein. In some embodiments, the fluorescent marker protein is GFP, ZsGreenl, mCherry, DsRed-Monomer, DsRed-Express, DsRed-Express2, tdTomato, AsRed2, mStrawberry, E2-Crimson, HcRedl, mRaspberry, mPlum, mOrange2, mBanana, ZsYellowl, Dendra2, PAmCherry, Timer, DsRed2, or AmCyanl. In any of the embodiments disclosed herein, the polynucleotide encoding the fluorescent marker protein can be operably linked to a promoter. In some embodiments, the promoter is an IE1 promoter or a 3xP3 promoter.

[0048] In any of the embodiments disclosed herein, the expression vector plasmid disclosed herein can further comprise an enhancer. In some embodiments, the enhancer is Hr5 enhancer. In any of the embodiments disclosed herein, the enhancer can be truncated.

[0049] In any of the embodiments disclosed herein, the expression vector plasmid can be introduced into the genome of an insect using piggyBac transposable elements, Mariner transposable elements, PhiC31, ZFNs, TALENs, or CRISPR.

[0050] In any of the embodiments disclosed herein, the expression vector plasmid can comprise a sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 1-17. In some embodiments, the expression vector plasmid comprises a sequence set forth in any one of SEQ ID NOs: 1-17.

[0051] In some embodiments, disclosed herein is a genetically engineered insect comprising a gene expression system incorporated into a chromosome of the insect, the gene expression system comprising: i. a doublesex (dsx) splice control module wherein the dsx splice control module comprises the components from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a first portion of a dsx exon 5 sequence; iv) an effector protein coding sequence; v) a second portion of a dsx exon 5 sequence; vi) a dsx intron 5; and vii) a dsx exon 6; and ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect.

[0052] In some embodiments, disclosed herein is a genetically engineered insect comprising a gene expression system incorporated into a chromosome of the insect, the gene expression system comprising: i. a doublesex (dsx) splice control module polynucleotide operably linked to an effector protein coding sequence, wherein the dsx splice control module comprises, from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a dsx exon 5 sequence; iv) a dsx intron 5 sequence; and v) a dsx exon 6 sequence; wherein the effector protein coding sequence is 3’ of the dsx splice control module polynucleotide; ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the effector protein coding sequence; and iv. a promoter operable in the insect.

[0053] In any of the embodiments disclosed herein, the dsx splice control module can be derived from an Anopheles dsx.

[0054] In some embodiments, disclosed herein is a genetically engineered insect comprising a gene expression system incorporated into a chromosome of the insect, the gene expression system comprising: a first module comprising from 5 ’ to 3 ’ : i. a promoter operable in the insect; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR; and a second module comprising from 5’ to 3’ : i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a female- specific lethal protein; and iv. a 3’ UTR.

[0055] In any of the embodiments disclosed herein, the insect can be a mosquito. In some embodiments, the mosquito is a mosquito of the genus Anopheles, Aedes, Stegomyia or Culex. In some embodiments, the mosquito is Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freeborni, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles arabiensis, Anopheles coluzzii, or Anopheles gambiae.

[0056] In some embodiments, disclosed herein is a female genetically engineered insect comprising a gene expression system incorporated into a chromosome of the insect, wherein the gene expression system comprises: i. a doublesex (dsx) splice control module wherein the splice control module is capable to produce a splice form comprising the components from 5’ to 3’: i) a dsx exon 4 sequence; iii) a first portion of a dsx exon 5 sequence; iv) an effector protein coding sequence; v) a second portion of a dsx exon 5 sequence; and vii) a dsx exon 6; wherein the dsx splice control module is spliced specifically in the female genetically engineered insect to produce one or more female splice forms that express the effector protein; and ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect.

[0057] In some embodiments, the effector protein is lethal, deleterious, or sterilizing to the insect.

[0058] In any of the embodiments disclosed herein, the gene expression system further can comprise a polynucleotide encoding a fluorescent protein. In some embodiments, the polynucleotide encoding the fluorescent protein is operably linked to a promoter. In some embodiments, the fluorescent protein is selected from the group consisting of GFP, ZsGreenl, mCherry, DsRed-Monomer, DsRed-Express, DsRed-Express2, tdTomato, AsRed2, mStrawberry, E2-Crimson, HcRedl, mRaspberry, mPlum, mOrange2, mBanana, ZsYellowl, Dendra2, PAmCherry, Timer, DsRed2, and AmCyanl, and the promoter is an IE1 promoter or a 3xP3 promoter.

[0059] In some embodiments, disclosed herein is a method of producing a genetically engineered insect comprising inserting a gene expression system into the insect’s genome, wherein the gene expression system comprises: i. a doublesex (dsx) splice control module wherein the splice control module comprises the components from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a first portion of a dsx exon 5 sequence; iv) an effector protein coding sequence; v) a second portion of a dsx exon 5 sequence; vi) a dsx intron 5; and vii) a dsx exon 6; and ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect; or wherein the gene expression system comprises: i. a doublesex (dsx) splice control module polynucleotide operably linked to an effector protein coding sequence, wherein the dsx splice control module comprises, from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a dsx exon 5 sequence; iv) a dsx intron 5 sequence; and v). a dsx exon 6 sequence; wherein the effector protein coding sequence is 3’ of the dsx splice control module polynucleotide; ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the effector protein coding sequence; and iv. a promoter operable in the insect. In some embodiments, the dsx splice control module is derived from an Anopheles dsx.

[0060] In some embodiments, disclosed herein is a method of producing a genetically engineered insect comprising inserting a gene expression system into the insect’s genome, wherein the gene expression system comprises: a first module comprising from 5’ to 3’: i. a promoter operable in the insect; ii. a coding sequence of tTAV or a variant thereof; iii. a 3’ UTR; and a second module comprising from 5’ to 3’: i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a female- specific lethal protein; and iv. a 3’ UTR.

[0061] In any of the embodiments disclosed herein, the insect can be a mosquito. In some embodiments, the mosquito is a mosquito of the genus Anopheles, Aedes, Stegomyia or Culex. In some embodiments, the mosquito is Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freeborni, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles arabiensis, Anopheles coluzzii, or Anopheles gambiae.

[0062] In some embodiments, the gene expression system further comprises a polynucleotide encoding a fluorescent protein. In some embodiments, the polynucleotide encoding the fluorescent protein is operably linked to a promoter. In some embodiments, the fluorescent protein is selected from the group consisting of GFP, ZsGreenl, mCherry, DsRed- Monomer, DsRed-Express, DsRed- Express!, tdTomato, AsRed2, mStrawberry, E2-Crimson, HcRedl, mRaspberry, mPlum, mOrange2, mBanana, ZsYellowl, Dendra2, PAmCherry, Timer, DsRed2, and AmCyanl, and the promoter is an IE1 promoter or a 3xP3 promoter.

[0063] In some embodiments, disclosed herein is a method of selectively rearing male genetically engineered insects comprising, rearing a genetically engineered insect disclosed herein, wherein the rearing is in the absence of tetracycline or an analogue thereof.

[0064] In some embodiments, disclosed herein is a male genetically engineered male insect produced by the method disclosed herein.

[0065] In some embodiments, disclosed herein is a method of reducing a wild insect population comprising contacting the wild insect population with a plurality of the male genetically engineered insects disclosed herein, wherein the male genetically engineered insects mate with wild female insects. In some embodiments, the insect is a mosquito. In some embodiments, the mosquito is a mosquito of the genus Anopheles, Aedes, Stegomyia or Culex. In some embodiments, the mosquito is Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freeborni, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles arabiensis, Anopheles coluzzii, or Anopheles gambiae.

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIGs. 1A-1C depict exemplary architectures of a doublesex (dsx) splice control module (top rows) operably linked to an effector protein coding gene. The middle rows depict female- specific dsx splice forms, and the bottom rows depict male-specific dsx splice forms, of the dsx splice control module above. Depicted components of the modules include dsx exon 4 or a portion thereof (Ex.4), dsx intron 4 or a portion thereof (Intron 4), dsx intron 5 or a portion thereof (Intron 5), dsx exon 5 or a portion thereof (Ex.5), dsx exon 6 or a portion thereof (Ex.6), and ubiquitin encoding polynucleotide (Ubi, an exemplary fusion leader sequence). tTAV is an exemplary effector protein coding gene. FIG. 1A depicts an exemplary architecture of a dsx splice control module operably linked to an effector protein coding gene (tTAV) wherein the effector protein coding gene is incorporated into the splice control module. FIG. IB depicts an exemplary architecture of a dsx splice control module operably linked to an effector protein coding gene wherein the effector protein coding gene is 3’ of the splice control module, and the splice control module forms a continuous open reading frame in females that includes the effector protein coding gene. FIG. 1C depicts an exemplary architecture of a dsx splice control module operably linked to an effector protein coding gene wherein the effector protein coding gene is 3’ of the splice control module, and the splice control module forms a continuous open reading frame in females that includes the effector protein coding gene.

[0067] FIGs. 2A-2Q depict exemplary recombinant DNA constructs comprising a dsx splice control module and a color marker cassette. The middle two rows depict female- specific dsx splice forms (Fl and F2), and the bottom rows depict male-specific dsx splice forms, of the dsx splice control module above. FIG. 2A depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2B depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2C depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2D depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2E depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2F depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2G depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2H depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 21 depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2J depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2K depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2L depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2M depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2N depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 20 depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2P depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette. FIG. 2Q depicts an exemplary recombinant DNA construct comprising a dsx splice control module and a color marker cassette.

[0068] FIGs. 3A-3C depict exemplary modifications of a dsx splice control module. FIG. 3A shows a schematic drawing of the alignment between the dsx splice control module comprised in construct 0X5680 and a wildtype Anopheles stephensi dsx showing the elements (exons as dark grey bars and introns as white bars) and the locations of the modifications shown as short dashes. FIG. 3B shows sequence alignment between the dsx splice control module and a wild type Anopheles stephensi dsx. The first row is the consensus sequence (SEQ ID NO: 70). The second row is the sequence in strain 0X5680 (SEQ ID NO: 71). The third row is wild type Anopheles stephensi dsx (SEQ ID NO: 72). FIG. 3C shows sequence alignment between the dsx splice control module and a wild type Anopheles albimanus dsx. The first row is the wild type Anopheles albimanus dsx (SEQ ID NO: 73). The second to fourth rows are the sequences in strain 0X5721 (SEQ ID NO: 74), 0X5734 (SEQ ID NO: 75), and 0X5733 (SEQ ID NO: 76), respectively. Green highlight denotes nucleotide changed to remove a MET (start) codon. Red highlight denotes nucleotide changed to remove a STOP codon. Cyan highlight denotes nucleotide changed to facilitate gene synthesis. Yellow highlight denotes Kozak & tTAV2 sequence.

[0069] FIGs. 4A-4B depict exemplary recombinant DNA constructs comprising a fist module comprising a coding sequence for tTAV or a variant thereof, a second module comprising a female- specific lethal gene linked to a promoter repressible by tetracycline or by a tetracycline analogue, and a color marker cassette. FIG. 4A depicts exemplary recombinant DNA constructs comprising a fist module comprising a coding sequence for tTAV or a variant thereof, a second module comprising a female- specific lethal gene linked to a promoter repressible by tetracycline or by a tetracycline analogue, and a color marker cassette, the female- specific lethal gene being YOB. FIG. 4B depicts exemplary recombinant DNA constructs comprising a fist module comprising a coding sequence for tTAV or a variant thereof, a second module comprising a female- specific lethal gene linked to a promoter repressible by tetracycline or by a tetracycline analogue, and a color marker cassette, the female- specific lethal gene being GUY1.

DETAILED DESCRIPTION OF THE INVENTION

[0070] In one aspect, disclosed herein is a doublesex (dsx') splice control module polynucleotide comprising, from 5’ to 3’: i. a dsx exon 4 sequence; ii. a dsx intron 4 sequence; iii. a first portion of a dsx exon 5 sequence ; iv. an effector protein coding sequence; v. a second portion of the dsx exon 5 sequence; vi. a dsx intron 5 sequence; and vii. a dsx exon 6 sequence.

[0071] In another aspect, disclosed herein is a doublesex (dsx) splice control module polynucleotide operably linked to an effector protein coding sequence, comprising, from 5’ to 3’: i. a dsx exon 4 sequence; ii. a dsx intron 4 sequence; iii. a dsx exon 5 sequence; iv. a dsx intron 5 sequence; and v. a dsx exon 6 sequence; wherein the effector protein coding sequence is 3’ of the dsx splice control module polynucleotide.

[0072] In another aspect, disclosed herein is a gene expression system comprising the dsx splice control module polynucleotide disclosed herein. [0073] In another aspect, disclosed herein is a gene expression polynucleotide, comprising a first module comprising from 5’ to 3’ : i. a promoter operable in an insect; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR; and a second module comprising from 5’ to 3’: i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a sex-specific lethal protein; and iv. a 3’ UTR.

[0074] In another aspect, disclosed herein is an expression vector plasmid comprising the gene expression system or the gene expression polynucleotide disclosed herein.

[0075] In another aspect, disclosed herein is a genetically engineered insect comprising a gene expression system incorporated into a chromosome of the insect, the gene expression system comprising: i. a doublesex (dsx) splice control module wherein the splice control module comprises the components from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a first portion of a dsx exon 5 sequence; iv) an effector protein coding sequence; v) a second portion of a dsx exon 5 sequence; vi) a dsx intron 5; and vii) a dsx exon 6; ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect.

[0076] In another aspect, disclosed herein is a female genetically engineered insect comprising a gene expression system incorporated into a chromosome of the insect, wherein the gene expression system comprises: i. a doublesex (dsx') splice control module wherein the splice control module is capable to produce a splice form comprising the components from 5’ to 3’: i) a dsx exon 4 sequence; iii) a first portion of a dsx exon 5 sequence comprising a 5’ terminal fragment of dsx exon 5; iv) an effector protein coding sequence, wherein the effector protein is lethal, deleterious sterilizing, or otherwise leading to a dosage sex determination bias to an insect; v) a second portion of a dsx exon 5 sequence comprising a 3’ terminal fragment of dsx exon 5; vii) a dsx exon 6; wherein the dsx splice control module is spliced specifically in the female genetically engineered insect to produce one or more female splice forms that express the effector protein; ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect.

[0077] In another aspect, disclosed herein is a genetically engineered insect comprising a gene expression system incorporated into a chromosome of the insect, the gene expression system comprising: i. a doublesex (dsx) splice control module polynucleotide operably linked to an effector protein coding sequence, wherein the dsx splice control module comprises, from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a dsx exon 5 sequence; iv) a dsx intron 5 sequence; and v) a dsx exon 6 sequence; wherein the effector protein coding sequence is 3’ of the dsx splice control module polynucleotide; ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the effector protein coding sequence; and iv. a promoter operable in the insect.

[0078] In another aspect, disclosed herein is a genetically engineered insect comprising a gene expression system incorporated into a chromosome of the insect, the gene expression system comprising a first module comprising from 5’ to 3’ : i. a promoter operable in an insect; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR; and a second module comprising from 5’ to 3’ : i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a sex-specific lethal protein; and iv. a 3’ UTR.

[0079] In another aspect, disclosed herein is a method of producing a genetically engineered insect comprising inserting a gene expression system into the insect’s genome, wherein the gene expression system comprises: i. a doublesex (dsx') splice control module wherein the splice control module comprises the components from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a first portion of a dsx exon 5 sequence; iv) an effector protein coding sequence; v) a second portion of a dsx exon 5 sequence; vi) a dsx intron 5; and vii) a dsx exon 6; ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect; or wherein the gene expression system comprises: i. a doublesex (dsx) splice control module polynucleotide operably linked to an effector protein coding sequence, wherein the dsx splice control module comprises, from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a dsx exon 5 sequence; iv) a dsx intron 5 sequence; and v) a dsx exon 6 sequence; wherein the effector protein coding sequence is 3’ of the dsx splice control module polynucleotide; ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the effector protein coding sequence; and iv. a promoter operable in the insect.

[0080] In another aspect, disclosed herein is a method of producing a genetically engineered insect comprising inserting a gene expression system into the insect’s genome, wherein the gene expression system comprises: a first module comprising from 5’ to 3’: i. a promoter operable in an insect; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR; and a second module comprising from 5’ to 3’: i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a sex-specific lethal protein; and iv. a 3’ UTR.

[0081] In another aspect, disclosed herein is a method of selectively rearing male genetically engineered insects comprising, rearing a genetically engineered insect disclosed herein, wherein the rearing is in the absence of a tetracycline analogue.

[0082] In another aspect, disclosed herein is a genetically engineered male insect produced by the method of selectively rearing male genetically engineered insects disclosed herein.

[0083] In yet another aspect, disclosed herein is a method of reducing a wild insect population comprising contacting the wild insect population with a plurality of the male genetically engineered insects disclosed herein, wherein the male genetically engineered insects mate with wild female insects.

I. Definitions

[0084] This description contains citations to various journal articles, patent applications and patents. These are herein incorporated by reference as if each was set forth herein in its entirety.

[0085] The term “penetrance,” as used herein, refers to the proportion of individuals carrying a particular variant of a gene that also express the phenotypic trait associated with that variant. Thus, “penetrance”, in relation to the present invention, refers to the proportion of transformed organisms which express the phenotypic trait.

[0086] The term “construct,” as used herein, refers to an artificially constructed segment of DNA for insertion into a host organism, for genetically modifying the host organism. At least a portion of the construct is inserted into the host organism's genome and alters the phenotype of the host organism. The construct may form part of a vector or be the vector.

[0087] The term “transgene,” as used herein, refers to the polynucleotide sequence comprising a gene expression system to be inserted into a host organism's genome, to alter the host organism's phenotype. The portion of the plasmid vector containing the genes to be expressed (as shown in FIGs. 1A-1C, for example) is referred to herein as the transfer DNA or recombinant DNA (rDNA). [0088] The term “gene expression system,” as used herein, refers to a gene to be expressed together with any genes and DNA sequences which are required for expression of said gene to be expressed.

[0089] A “splice control module polynucleotide”, when introduced into an insect as a part of a vector, undergoes differential splicing (e.g., stage- specific, sex-specific, tissue-specific, germline- specific, etc.) together with a spliceosome, and thus creates different transcripts. If the splice control module is spliced in a sex- specific manner, different transcripts are created in females than males. A “splice control module polynucleotide” may contain multiple splice control sequences that join multiple exons to form a polypeptide encoding nucleic acid.

[0090] The term “transactivation activity,” as used herein, refers to the activity of an activating transcription factor, which results in an increased expression of a gene. The activating transcription factor may bind a promoter or operator operably linked to said gene, thereby activating the promoter and, consequently, enhancing the expression of said gene. Alternatively, the activating transcription factor may bind an enhancer associated with said promoter, thereby promoting the activity of said promoter via said enhancer.

[0091] The term “lethal gene,” as used herein, refers to a gene whose expression product has, when expressed in sufficient quantity, a lethal effect, on the organism within which the lethal gene is expressed.

[0092] The term “lethal effect,” as used herein, refers to a deleterious or sterilizing effect, such as an effect capable of killing the organism per se or its offspring, or capable of reducing or destroying the function of certain tissues thereof, of which the reproductive tissues are particularly preferred, so that the organism or its offspring are sterile. Therefore, some lethal effects, such as poisons, will kill the organism or tissue in a short time-frame relative to their life-span, whilst others may simply reduce the organism's ability to function, for instance reproductively. The term also encompasses dosage sex determination biases.

[0093] The term “tTAV gene variant,” as used herein, refers to a polynucleotide encoding the functional tetracycline repressible Trans-Activator (tTA) protein but which differ in the sequence of nucleotides. These nucleotides may encode different tTA protein sequences, such as, for example, tTAV2 and tTAV3. [0094] The term “promoter,” as used herein, refers to a DNA sequence, generally directly upstream to the coding sequence, required for basal and/or regulated transcription of a gene. In particular, a promoter is sufficient to allow initiation of transcription, generally having a transcription initiation start site and a binding site for the RNA polymerase transcription complex.

[0095] The term “minimal promoter” (also referenced herein as “minipromoter” and “minipro”) as used herein, refers to a promoter as defined above, generally having a transcription initiation start site and a binding site for the polymerase complex, and further generally having sufficient additional sequence to permit these two to be effective. Other sequences, such as that which determines tissue specificity, for example, may be lacking.

[0096] The term “exogenous control factor,” as used herein, refers to a substance which is not found naturally in the host organism and which is not found in a host organism's natural habitat, or an environmental condition not found in a host organism's natural habitat. Thus, the presence of the exogenous control factor is controlled by the manipulator of a transformed host organism in order to control expression of the gene expression system.

[0097] The term “tetO element,” as used herein, refers to one or more tetO operator units positioned in series. The term, for example, “tetOx(number),” as used herein, refers to a tetO element consisting of the indicated number of tetO operator units. For example, references to “tetOx7” indicates a tetO element consisting of 7 tetO operator units. Similarly, references to “tetOxl4” refers to a tetO element consisting of 14 tetO operator units, and so on.

[0098] Where reference to a particular nucleotide or protein sequence is made, it will be understood that this includes reference to any mutant or variant thereof, having substantially equivalent biological activity thereto. Preferably, the mutant or variant has at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99%, preferably at least 99.9%, and most preferably at least 99.99% sequence identity with the reference sequences.

[0099] However, it will be understood that despite the above sequence homology, certain elements, in particular the flanking nucleotides and splice branch site must be retained, for efficient functioning of the system. In other words, whilst portions may be deleted or otherwise altered, alternative splicing functionality or activity, to at least 30%, preferably 50%, preferably 70%, more preferably 90%, and most preferably 95% compared to the wild type should be retained. This could be increased compared to the wild type, as well, by suitably engineering the sites that bind alternative splicing factors or interact with the spliceosome, for instance.

[0100] As used herein, doublesex (dsx) refers to a gene in both male and female insects, such as Anopheles, that is subject to alternative splicing.

[0101] As used herein, “5’ UTR,” refers to an untranslated region of an RNA transcript that is 5’ of the translated portion of the transcript and often contains a promoter sequence.

[0102] As used herein, “3’ UTR,” refers to an untranslated region of an RNA transcript that is 3’ of the translated portion of the transcript and often contains a polyadenylation sequence.

[0103] As used herein, “a portion” of a sequence refers to less than or equal to a full length sequence.

II. Overview of the Technology

[0104] The invention provides constructs and methods for sex- selecting insects via 1) differentially expressing proteins in insects in a sex- specific manner such that either a male insect or a female insect will express the protein in significant quantities and the other will not, or 2) expressing proteins that are specifically lethal to one sex and not the other. Some embodiments of the constructs of the invention have been engineered with a splice control module that is spliced differently in male insects compared to female insects. The splice control module may be operably linked to a heterologous protein-encoding polynucleotide such that the heterologous protein of interest is expressed in a sex-specific manner when introduced into an insect species. Some embodiments of the constructs of the invention have been engineered with a female-lethal protein expression cassette. The expression cassette may be similarly expressed in male and female insects, but the protein product is specifically lethal to female insects. The constructs of the invention also may contain other elements for regulating expression in an insect, for identifying insects that have an integrated construct in their genome, and for selecting transformed cells and/or insects, for example as will be described more fully below. A. Sex-Specific Self-Limiting Via Splice Control

1. The Splice Control Module

[0105] Thus, in a first aspect, the present invention provides a splice control module polynucleotide which provides for differential splicing (e.g., sex-specific, stage-specific, germline- specific, tissue-specific, etc.) in an organism. In particular, the invention provides a splice control module which provides for sufficient female- specificity of the expression of a gene of interest to be useful. In certain embodiments of the invention, the gene of interest is a lethal gene that imparts a deleterious, lethal, sterilizing effect, or otherwise leading to a dosage sex determination bias. For convenience, the description will refer to a lethal effect, however, it will be understood that the splice module may be used on other genes of interest as described in further detail below.

[0106] Expression of the dominant lethal genes may be sex-specific, or be a combination of sex-specific and stage-specific, germline- specific or tissue-specific, due to the presence of at least one splice control module in each gene expression system operably linked to a gene of interest to be differentially expressed. In some embodiments, the sex-specific expression is female- specific. The splice control module in each gene expression sequence allows an additional level of control of protein expression, in addition to the promoter.

[0107] The gene from which the splice control module is derived from may comprise a coding sequence for a protein or polypeptide, i.e., at least two exons, capable of encoding a polypeptide, such as a protein or fragment thereof. Preferably, the different exons are differentially spliced together to provide alternative mRNAs. Preferably, said alternative spliced mRNAs have different coding potential, i.e., encode different proteins or polypeptide sequences. Thus, the expression of the coding sequence is regulated by alternative splicing.

[0108] Each splice control module in the system comprises at least one splice acceptor site and at least one splice donor site. The number of donor and acceptor sites may vary, depending on the number of segments of sequence that are to be spliced together.

[0109] In some embodiments, the splice control module regulates the alternative splicing by means of both intronic and exonic nucleotides. It will be understood that in alternative splicing, sequences may be intronic under some circumstances (i.e., in some alternative splicing variants where introns are spliced out), but exonic under other. In other embodiments, the splice control module is an intronic splice control module. In other words, it is preferred that said splice control sequence is substantially derived from polynucleotides that form part of an intron and are thus excised from the primary transcript by splicing, such that these nucleotides are not retained in the mature mRNA sequence.

[0110] As mentioned above, exonic sequences may be involved in the mediation of the control of alternative splicing, but it is preferred that at least some intronic control sequences are involved in the mediation of the alternative splicing.

[0111] The splice control module may be removed from the pre-RNA, by splicing or retained so as to encode a fusion protein of at least a portion of the gene of interest to be differentially expressed.

[0112] Interaction of the splice control module with cellular splicing machinery, e.g., the spliceosome, leads to or mediates the removal of a series of, preferably, at least 50 consecutive nucleotides from the primary transcript and ligation (splicing) together of nucleotide sequences that were not consecutive in the primary transcript (because they, or their complement if the antisense sequence is considered, were not consecutive in the original template sequence from which the primary transcript was transcribed). Said series of at least 50 consecutive nucleotides comprises an intron. This mediation acts preferably in a sex-specific, more preferably, femalespecific, manner such that equivalent primary transcripts in different sexes, and optionally also in different stages, tissue types, etc., tend to remove introns of different size or sequence, or in some cases may remove an intron in one case but not another. This phenomenon, the removal of introns of different size or sequence in different circumstances, or the differential removal of introns of a given size or sequence, in different circumstances, is known as alternative splicing. Alternative splicing is a well-known phenomenon in nature, and many instances are known.

[0113] Where mediation of alternative splicing is sex-specific, it is preferred that the splice variant encoding a functional protein to be expressed in an organism is the Fl splice variant, i.e., a splice variant where the F denotes it is found only or predominantly in females, although this is not essential.

[0114] When exonic nucleotides are to be removed, then these must be removed in multiples of three (entire codons), if it is desired to avoid a frameshift, but as a single nucleotide or multiples of two (that are not also multiples of three) if it is desired to induce a frameshift. It will be appreciated that if only one or certain multiples of two nucleotides are removed, then this could lead to a completely different protein sequence being encoded at or around the splice junction of the mRNA.

[0115] Correspondingly for configurations where all or part of a functional open reading frame is on a cassette exon, it is preferred that this cassette exon is included in transcripts found only or predominantly in females, and preferably such transcripts are, individually or in combination, the most abundant variants found in females, although this is not essential.

[0116] In some embodiments, sequences are included in a hybrid or recombinant sequence or construct which are derived from naturally occurring intronic sequences which are themselves subject to alternative splicing, in their native or original context. Therefore, an intronic sequence may be considered as one that forms part of an intron in at least one alternative splicing variant of the natural analogue. Thus, sequences corresponding to single contiguous stretches of naturally occurring intronic sequence are envisioned, but also hybrids of such sequences, including hybrids from two different naturally occurring intronic sequences, and also sequences with deletions or insertions relative to single contiguous stretches of naturally occurring intronic sequence, and hybrids thereof. Said sequences derived from naturally occurring intronic sequences may themselves be associated, in the invention, with sequences not themselves part of any naturally occurring intron. If such sequences are transcribed, and preferably retained in the mature RNA in at least one splice variant, they may then be considered exonic.

[0117] In some embodiments, the splice control module comprises one or more modifications such as substitutions, insertions, and deletions compared to a wildtype sequence from which the splice control module is derived. In some embodiments, the splice control module comprises one or more modifications to remove existing or potential open reading frames. This may be preferable when a gene of interest to be expressed is part of the splice control module and it is desired to start translation at the gene of interest to avoid translating part of the upstream splice control module. For example, modifications to the splice control module are illustrated in FIGs. 3A-3C.

[0118] Production from different splice variants of two or more different proteins or polypeptide sequences of differential function is also envisioned, in addition to the production of two or more different proteins or polypeptide sequences of which one or more has no predicted or discernable function. Also envisioned is the production from different splice variants of two or more different proteins or polypeptide sequences of similar function, but differing subcellular location, stability or capacity to bind to or associate with other proteins or nucleic acids.

2. Examples of Splice Control Modules a. Doublesex (dsx)

[0119] In some embodiments, the splice control module polynucleotide comprises at least a fragment of the doublesex (dsx) gene derived from an arthropod, such as a culicid. In some embodiments, more than one splice control module polynucleotide is derived from dsx, and the dsx genes are derived from the same or different species. In some embodiments, the dsx gene is derived from a species of the Order Diptera, such as, but not limited to those of the genus Anopheles, Aedes, Cochliomyia, Culex, Drosophila, Glossina, Lucilia, Lutzomyia, Ceratitis, Bactrocera, Anastrepha, Mayetiola, Megaselia, Musca, Phlebotomus, and Rhagoletis. In some embodiments, the splice control module polynucleotide is derived from an Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles spp. , Anopheles gambiae, Aedes aegypti, Anastrepha spp., Ceratitis capitata, Bactrocera oleae, Bactrocera dorsalis, Bactrocera zonata, Bactrocera correcta, Bactrocera tryoni, Ceratitis rosa, Cochliomyia homnivorax, Cochliomyia macellaria, Culex quinquefasciatus, Drosophila Americana, Drosophila erecta, Drosophila hydei, Drosophila mauritania, Drosophila melanogaster, Drosophila sechellia, Drosophila simulans, Drosophila virilis, Glossina morsitans, Lucilia cuprina, Lucilia sericata, Lutzomyia longipalpis, Mayetiola destructor, Megaselia scalaris, Musca domestica, or Phlebotomus papatasi dsx.

[0120] In embodiments wherein more than one splice control sequence is derived from dsx, the splice control sequences may be derived from the same species. In other embodiments, the splice control sequences are derived from different species. In some embodiments, the splice control sequences are derived from the same insect, such as, for example, Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles spp., or Anopheles gambiae.

[0121] The dsx splice control module allows a sex-specific splicing of the module to a polypeptide encoding polynucleotide such that the polypeptide is expressed in a sex-specific manner.

[0122] In some embodiments, the present invention provides a doublesex dsx) splice control module polynucleotide wherein the splice control module comprises (from 5’ to 3’): a dsx exon 4 sequence, a dsx intron 4 sequence, a first portion of a dsx exon 5 sequence, an effector protein coding sequence (such as, for instance, SEQ ID NO: 18), a second portion of a dsx exon 5 sequence, a dsx intron 5 sequence, and a dsx exon 6 sequence. An example of such a dsx splice control module polynucleotide is shown in FIG. 1A.

[0123] In some embodiments, the dsx exon 4 sequence is a wildtype dsx exon 4. In some embodiments, the dsx exon 4 sequence is a modified dsx exon 4. In some embodiments, the dsx exon 4 sequence is modified compared to a wild-type dsx exon 4 sequence to remove one or more start codons. In some embodiments, the dsx exon 4 sequence is modified compared to a wild- type dsx exon 4 sequence to remove one or more stop codons.

[0124] Removing start codons and/or stop codons could be preferable when it is desired to remove open reading frames. In some embodiments, the dsx exon 4 sequence is a modified dsx exon 4 to remove one or more existing open reading frame that is 5’ of the effector protein coding sequence. In some embodiments, the dsx exon 4 sequence is a modified dsx exon 4 to remove one or more potential open reading frames 5’ of the effector protein coding sequence. In some embodiments, the modified dsx exon 4 comprises one or more modifications selected from the group consisting of a substitution, an insertion, and a deletion.

[0125] In some embodiments, the first portion of the dsx exon 5 sequence is a wildtype dsx exon 5. In some embodiments, the first portion of the dsx exon 5 sequence is a portion (such as a 5’ terminal fragment) of a wildtype dsx exon 5. In some embodiments, the first portion of the dsx exon 5 sequence is modified compared to a wild-type dsx exon 5 sequence to remove one or more start codons. In some embodiments, the dsx exon 5 sequence is modified compared to a wild-type dsx exon 5 sequence to remove one or more stop codons.

[0126] In some embodiments, the dsx exon 5 sequence is a modified dsx exon 5 to remove one or more existing open reading frame that is 5’ of the effector protein coding sequence. In some embodiments, the dsx exon 5 sequence is a modified dsx exon 5 to remove one or more potential open reading frames 5’ of the effector protein coding sequence. In some embodiments, the modified dsx exon 5 comprises one or more modifications selected from the group consisting of: a substitution, an insertion, and a deletion.

[0127] In some embodiments, the dsx exon 6 sequence is a wildtype dsx exon 6. In some embodiments, the dsx exon 6 sequence is a truncated dsx exon 6 to reduce the size of the splice control module polynucleotide.

[0128] An example of a modified dsx exon 4 and modified dsx exon 5 is shown in FIGs. 3B- 3C.

[0129] In some embodiment, two principle transcripts (Fl and F2) are made in female insects: Transcript Fl comprises the dsx exon 4 sequence, the first portion of a dsx exon 5 sequence, the effector protein coding sequence, the second portion of a dsx exon 5 sequence, and the dsx exon 6 sequence; Transcript F2 comprises the same components, while the length of the first portion of the dsx exon 5 sequence is shorter in Transcript F2. In male insects, the splice form contains the dsx exon 4 sequence and the exon 6 sequence. Examples of such sexspecific splicing patterns are illustrated in FIGs. 2A-2Q.

[0130] In some embodiments, the effector protein coding sequence comprises a start codon at the 5’ end. In some embodiments, the 5’ end of the effector protein coding sequence is 3’ to a start codon. In some embodiments, the effector protein coding sequence comprises an inframe stop codon at the 3’ end. In some embodiments, the effector protein coding sequence is 5’ to the first in-frame stop codon. In some embodiments, the in-frame stop codon is the first in-frame stop codon (that is 3’ to and in frame with the start codon). Since the effector protein coding sequence is retained (spliced-in) in the female transcript forms and is within the open reading frame defined by the start codon and the first in-frame stop codon, the effector protein is expressed in female insects.

[0131] In some embodiments, the dsx exon 4 sequence and/or the first portion of the dsx exon 5 sequence comprise at least one substitution, insertion, and/or deletion to remove any open reading frame through the entire exon 4 and the first portion of the dsx exon 5 sequence. In such embodiments, the effector protein coding sequence comprises a start codon at the 5’ end and therefore translation only starts at the effector protein coding sequence in female insects, avoiding the translation of the splice control module 5’ of the effector protein coding sequence.

[0132] In some embodiments, Fl and F2 transcripts are produced at different levels in female insects. In some embodiments, Fl is the primary transcript in female insects (e.g., Fl is produced at a higher level than F2). In some embodiments, F2 is the primary transcript in female insects (e.g., F2 is produced at a higher level than Fl). In some embodiments, Fl and F2 transcripts are produced at the same level in female insects.

[0133] In some embodiments, the present disclosure provides a doublesex (dsx') splice control module polynucleotide operably linked to an effector protein coding sequence wherein the splice control module polynucleotide comprises (from 5’ to 3’): a dsx exon 4 sequence, a dsx intron 4 sequence, a dsx exon 5 sequence, a dsx intron 5 sequence, and a dsx exon 6 sequence. In some embodiments, the effector protein coding sequence is 3’ of the dsx splice control module polynucleotide.

[0134] In such an embodiment, two principle transcripts (Fl and F2) are made in female insects: Transcript Fl comprises the dsx exon 4 sequence, the first portion of a dsx exon 5 sequence, the effector protein coding sequence, the second portion of a dsx exon 5 sequence, and the dsx exon 6 sequence; Transcript F2 comprises the same components, while the length of the first portion of the dsx exon 5 sequence is shorter in Transcript F2. The female splice forms also comprise the effector protein coding sequence in frame with the rest of the transcript. The start codon might be comprised by the splice control module polynucleotide, or the splice control module polynucleotide is 3’ to the start codon. The stop codon is preferably at the 3’ end of the effector protein coding sequence and in frame with the start codon in female insects. Thus the Fl and F2 transcripts are able to be translated to produce the effector protein. In male insects, the transcript contains the exon 4 sequence, the exon 6 sequence, and the effector protein coding sequence. However, the open reading frame ends before the effector protein coding sequence, i.e., the first in-frame stop codon is 5’ to the effector protein coding sequence. Such splicing patterns are illustrated in FIGs. 1B-1C.

[0135] In some embodiments, a dsx exon 4 sequence comprises the full length sequence of the corresponding dsx exon 4 in wild type Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles spp., or Anopheles gambiae.

[0136] In some embodiments, a dsx intron 4 sequence comprises the full length sequence of the corresponding dsx intron 4 sequence in wild type Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles spp., or Anopheles gambiae.

[0137] In some embodiments, a first portion of a dsx exon 5 sequence comprises a 5’ terminal fragment of dsx exon 5 in wild type Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles spp., or Anopheles gambiae dsx. In some embodiments, a first portion of a dsx exon 5 sequence comprises a full length wild type sequence. In some embodiments, a first portion of a dsx exon 5 sequence comprises less than a full length wild type sequence.

[0138] In some embodiments, a second portion of a dsx exon 5 sequence comprises a 3’ terminal fragment of dsx exon 5 in wild type Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles spp., or Anopheles gambiae dsx.

[0139] In some embodiments, a dsx intron 5 sequence comprises the full length sequence of the corresponding dsx intron 5 in wild type Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles spp., or Anopheles gambiae dsx. [0140] In some embodiments, a dsx exon 6 sequence comprises the full length sequence of the corresponding dsx exon 6 in wild type Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles spp., or Anopheles gambiae dsx. In some embodiments, the a dsx exon 6 sequence comprises a truncated corresponding dsx exon 6 in wild type dsx in any of the aforementioned species.

[0141] In some embodiments, one or more of the components, such as the dsx exon 4 sequence, the dsx intron 4 sequence, the first portion of the dsx exon 5 sequence, the second portion of the dsx exon 5 sequence, the dsx intron 5 sequence, and/or the dsx exon 6 sequence, comprise one or more modifications such as substitutions, insertions, and deletions, when compared to the corresponding sequence in, for instance, wild type Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles spp., or Anopheles gambiae. For example, in some embodiments, the dsx exon 4 sequence and the first portion of the dsx exon 5 sequence comprise one or more modifications such as substitutions, insertions, and deletions, when compared to the corresponding sequence in wild type Anopheles stephensi (FIG. 3B). In some embodiments, the dsx exon 4 sequence and the first portion of the dsx exon 5 sequence comprise one or more modifications such as substitutions, insertions, and deletions, when compared to the corresponding sequence in wild type Anopheles albimanus (FIG. 3C)

[0142] For instance, in some embodiments, the dsx exon 4 sequence is a portion of or is otherwise modified from a wild type exon 4 sequence in any of the aforementioned species. In some embodiments, the dsx intron 4 sequence is a portion of or is otherwise modified from a wild type intron 4 sequence in any of the aforementioned species. In some embodiments, the first portion of the dsx exon 5 sequence is a portion of or is otherwise modified from a wild type dsx exon 5 in any of the aforementioned species. In some embodiments, the first portion of the dsx exon 5 sequence is a 5’ terminal fragment of a wild type dsx exon 5 in any of the aforementioned species. In some embodiments, the second portion of the dsx exon 5 sequence is a portion of or is otherwise modified from a wild type dsx exon 5 in any of the aforementioned species. In some embodiments, the second portion of the dsx exon 5 sequence is a 3’ terminal fragment of a wild type dsx exon 5 in any of the aforementioned species. In some embodiments, the dsx intron 5 sequence is a portion of or is otherwise modified from a wild type dsx intron 5 sequence in any of the aforementioned species. In some embodiments, the dsx exon 6 sequence is a portion of or is otherwise modified from a wild type dsx exon 6 sequence in any of the aforementioned species.

[0143] In some embodiments, the dsx intron 4 sequence is a truncated dsx intron 4 sequence compared to the dsx intron 4 sequence in any of the aforementioned species. In some embodiments, the dsx intron 4 sequence comprises at least a 5’ terminal fragment of the dsx intron 4 that contains at least a portion of the 5’ end of intron 4 and a 3’ fragment of the dsx intron 4 that contains at least a portion of the 3’ end of intron 4. In some embodiments, the dsx intron 5 sequence is a truncated dsx intron 5 sequence compared to the dsx intron 5 sequence in any of the aforementioned species. In some embodiments, the dsx intron 5 sequence comprises at least a 5’ terminal fragment of the dsx intron 5 that contains at least a portion of the 5’ end of intron 5 and a 3’ fragment of the dsx intron 5 that contains at least a portion of the 3’ end of intron 5.

[0144] Therefore in some embodiments, the present invention provides a doublesex (dsx) splice control module polynucleotide wherein the splice control module comprises (from 5’ to 3’): a modified dsx exon 4 sequence, a dsx intron 4 sequence, a modified first portion of a dsx exon 5 sequence comprising a 5’ terminal fragment of dsx exon 5, an effector protein coding sequence (such as, for instance, SEQ ID NO: 18), a second portion of a dsx exon 5 sequence comprising a 3’ terminal fragment of dsx exon 5, a dsx intron 5 sequence, and a truncated dsx exon 6 sequence. b. Transformer (tra)

[0145] As mentioned above, in some embodiments the manner or mechanism of alternative splicing is sex-specific, preferably female- specific, and any suitable splice control sequence may be used. In some embodiments, the splice control module is derived from a tra intron. The Ceratitis capitata tra intron from the transformer gene was initially characterized by Pane et al. (2002) Development 129:3715-3725. In insects, for instance, the tra protein is differentially expressed in different sexes. In particular, the tra protein is known to be present largely in females and, therefore, mediates alternative splicing in such a way that a coding sequence is expressed in a sex-specific manner, i.e., that in some cases a protein is expressed only in females or at a much higher level in females than in males or, alternatively, in other cases a protein is expressed only in males, or at a much higher level in males than in females. The mechanism for achieving this sex-specific alternative splicing mediated by the tra protein or the TRA/TRA-2 complex is known and is discussed, for instance, in Pane et al. (2002) Development 129:3715-3725.

[0146] It will be appreciated that homologues of the Ceratitis capitata tra intron from the transformer gene exist in other species, and these can be easily identified in said species and also in their various genera. Thus, when reference is made to tra it will be appreciated that this also relates to tra homologues in other species. Thus, in some embodiments each of the alternative splicing mechanisms is independently derived from the Ceratitis capitata tra intron (Cctra), or from another ortholog or homolog. In some embodiments, the ortholog or homologue is from an arthropod, such as an insect of the order Diptera, such as a tephritid. In other embodiments, the ortholog or homologue is from the genus Cochliomyia, Glossina, Lucilia, Musca, Ceratitis, Bactrocera, Anastrepha or Rhagoletis. In other embodiments, the ortholog or homolog is from Ceratitis rosa, or Bactrovera zonata. In further embodiments, the ortholog or homolog is from B. zonata, and this ortholog or homolog is referred to herein as Bztra (GenBank accession number BzTra KJ397268). Orthologs may also be from the Order Hymenoptera, or Coleoptera. Examples, include, but are not limited to Apis cerana, Apis dorsata, Apis florea, Apis mellifera, Alta cephalotes, Bombus impatiens, Bombus terrestris, Camponotus floridanus, Euglossa hemichlora, Harpegnathos saltator, Linepithema humile, Melipona compressipes, Megachile rotundata, Nasonia giraulti, Nasonia longicornis, Nasonia vitripennis, Pogonomyrmex barbatus, Solenopsis invicta, and Tribolium castaneum.

[0147] The splicing pattern in Cctra in particular is well conserved. Transcripts found in males contain additional exonic material relative to the Fl transcript, such that these transcripts do not encode full-length, functional tra protein. By contrast, the Fl transcript does encode full- length, functional tra protein; this transcript is substantially female- specific at most life-cycle stages, though it is speculated that very early embryos of both sexes may contain a small amount of this transcript.

[0148] The tra gene is regulated in part by sex- specific alternative splicing, while its key product, the tra protein, is itself involved in alternative splicing. In insects, sex-specific alternative splicing is mediated by the tra protein, or a complex comprising the tra and TRA2 proteins, including Dipteran splice control sequences derived from the doublesex (dsx) gene and also the tra intron itself, although this would exclude the tra intron from Drosophila (Dmtra'), which is principally mediated by the Sxl gene product in Drosophila, rather than tra or the TRA/TRA2 complex. Outside of Drosophila, the Sxl gene product is not differentially expressed in the different sexes. Sxl is not thought to act in the mediation of sex-specific alternative splicing in non-Drosophilid insects.

[0149] By ‘ ‘derived” it will be understood that, using reference to the tra intron, this refers to sequences that approximate to or replicate exactly the tra intron, as described in the art, in this case by Pane et al. (2002), supra. However, it will be appreciated that, as these are intronic sequences, that some nucleotides can be added or deleted or substituted without a substantial loss in function.

[0150] If more than one splice control module is incorporated into a gene expression system of the invention, the splice control modules may be the same or different. In some embodiments, it is preferred that the splice control modules are derived from different species in order to reduce the risk of recombination. Thus, in some embodiments, one of the splice control modules is derived from Cctra and the other is derived from a different species. For example, one of the splice control modules could be Cctra and the other could be Bztra (GenBank accession number BzTra KJ397268). The exact length of the splice control sequence derived from the tra intron is not essential, provided that it is capable of mediating alternative splicing. In this regard, it is thought that around 55 to 60 nucleotides is the minimum length for a modified tra intron. c. Actin-4

[0151] In other embodiments, the splice control module could be derived from the alternative splicing mechanism of the Actin-4 gene derived from an arthropod, preferably a tephritid. In some embodiments, the Actin-4 gene splice control module is derived from a species of the Ceratitis, the Bactrocera, the Anastrepha or the Rhagoletis genera. In some embodiments, the Actin-4 splice control module is derived from Ceratitis capitata, Bactrocera oleae, Ceratitis rosa, or Bactrocera zonata. In some embodiments, the Actin-4 splice control module is derived from Ceratitis capitata. In embodiments wherein more than one splice control module is derived from Actin-4, the splice control modules may be derived from the same or different species. d. Fruitless (fru)

[0152] In some embodiments, the splice control module could be derived from a fruitless (fru) gene. By a complex coordination of multiple promoters and alternative splicing, fru encodes proteins of the BTB-Zn finger (BTB-ZnF) family of transcription factors, and is spliced on a sex- specific basis. In some embodiments, the fru splice control module is derived from an Anopheles species. In embodiments wherein more than one splice control module is derived from/ru, the splice control modules may be derived from the same or different species.

[0153] While in some embodiments it is envisaged that there are more than one splice control module, which could be derived from the same gene or intron of origin, in other embodiments, there are more than one splice control module that may be derived from different genes or introns of origin. For example, in some embodiments, one of the splice control modules is derived from the tra intron and the other splice control module is derived from the Actin-4 gene or the dsx gene.

3. Splicing

[0154] Introns typically consist of the following features (given here as the sense DNA sequence 5’ to 3’); in RNAs, thymine (T) will be replaced by uracil (U)):

[0155] a. 5’ end (known as the splice “donor”): GT (or possibly GC)

[0156] b. 3’ end (known as the splice “acceptor”): AG [0157] c. A “branch point”, located anywhere from 18 to 40 nucleotides upstream from the 3' end of an intron. The branch point always contains an adenine, but it is otherwise loosely conserved. A typical sequence is YNYYRAY, where Y indicates a pyrimidine, N denotes any nucleotide, R denotes any purine, and A denotes adenine. During splicing, the adenine in the branch point initiates a nucleophilic attack on the 5' donor splice site. The free end of the upstream exon then initiates a second nucleophilic attack on the 3' acceptor splice site, releasing the intron as an RNA lariat and covalently combining the two exons. Preferably, branch points are included in each splice control sequence

[0158] The sequences provided can tolerate some sequence variation and still splice correctly. There are a few nucleotides known to be important. These are the ones required for all splicing. The initial GU and the final AG of the intron are particularly important, though ~5% of introns start GC instead. This consensus sequence is preferred, although it applies to all splicing, not specifically to alternative splicing.

[0159] The terminal nucleotides of exons immediately adjacent to the 5’ intro nic splice “donor” and the 3’ intronic splice “acceptor” are typically G. In some embodiments, the splice control module is immediately adjacent, in the 3’ direction, the start codon, so that the G of the ATG is 5’ to the start (5’ end) of the splice control module. This may be advantageous as it allows the G of the ATG start codon to be the 5’ G flanking sequence to the splice control module. For example, in an embodiment comprising (from 5’ to 3’): a dsx exon 4 sequence, a dsx intron 4 sequence, a dsx exon 5 sequence, a dsx intron 5 sequence, and a dsx exon 6 sequence, the splice control module is immediately adjacent, in the 3’ direction, the start codon.

[0160] In some embodiments, the splice control module polynucleotide is spliced on a sexspecific basis when introduced into an insect to produce one or more female splice forms in female insects and one or more male splice forms in male insects. In some embodiments, the one or more female splice forms comprise, from 5’ to 3’: i. a dsx exon 4 sequence; ii. a first portion of a dsx exon 5 sequence; iii. an effector protein coding sequence; iv. a second portion of a dsx exon 5 sequence; and vi. a dsx exon 6 sequence, the splice control module is capable of producing a plurality of different female splice forms in female insects, and wherein the length of the first portion of the dsx exon 5 sequence is variable between the plurality of different female splice forms, such as illustrated in FIGs. 2A-2Q. [0161] In some embodiments, the female splice forms express the effector protein. In some embodiments, the male splice form expresses the effector protein. In some embodiments, the male splice form does not express the effector protein in significant quantities. Not expressing the effector protein in significant quantities may include, for example, embodiments in which the male splice form does not express the effector protein at or above the limit of detection. In some embodiments, not expressing the effector protein in significant quantities comprises the male splice form expressing the effector protein at less than 0.01% of the expression level in females. In some embodiments, not expressing the effector protein in significant quantities comprises the male splice form expressing the effector protein at less than 0.1% of the expression level in females. In some embodiments, not expressing the effector protein in significant quantities comprises the male splice form expressing the effector protein at less than 1% of the expression level in females. In some embodiments, not expressing the effector protein in significant quantities comprises the male splice form expressing the effector protein at less than 10%, or 20%, or 50% of the expression level in females.

B. Sex-Specific Self-Limiting Via Sex-Specific Lethal Genes

[0162] The present invention also provides systems and methods for generating sex-specific self-limiting organisms using sex- specific lethal genes.

[0163] In one aspect, the present invention provides a gene expression polynucleotide, comprising a first module comprising from 5’ to 3’: i. a promoter operable in an insect; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR; and a second module comprising from 5’ to 3’: i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a sex-specific lethal protein; and iv. a 3’ UTR. In some embodiments, the sex-specific lethal protein is a female- specific lethal protein. In some embodiments, the female- specific lethal protein is GUY1 or a variant thereof. In some embodiments, the female-specific lethal protein is YOB or a variant thereof.

[0164] In some embodiments, by putting the expression of tTAV or a variant thereof under the control of bZipl, which is an early zygotic promoter, tTAV expression from very early embryonic development is achieved. Subsequently, in the absence of tetracyclines, tTAV can bind to the promoter of the second module, such as a TRE3G promoter, and stimulate expression of the female- specific lethal protein. C. Heterologous Sequences

[0165] The system is capable of expressing at least one heterologous gene of interest (such as an effector protein coding sequence and/or a female- specific lethal protein coding sequence) to make at least one gene product (e.g., a protein) of interest, such as a functional protein to be expressed in an organism (e.g., an effector protein or a female- specific lethal protein). The gene product (e.g., a protein) of interest may be, for example, lethal, deleterious, or sterilizing. In some embodiments, the gene product (e.g., a protein) of interest may be lethal, deleterious, or sterilizing to one sex (e.g., female) but not the other (e.g. male). In some embodiments, the gene product of interest may be a protein that has a therapeutic effect, or a marker (for instance DsRed2, AmCyanl, Green Fluorescent Protein (GFP), or one or more of their mutants or variants), or other markers that are well known in the art, such as drug resistance genes. Further proteins to be expressed in the organism are envisaged in combination with said functional protein, preferably a lethal gene as discussed below. Alternatively, a heterologous gene of interest may encode an RNA molecule that has a functional effect.

[0166] It is preferred that the expression of the one or more heterologous genes of interest leads to a phenotypic consequence in the organism. In some embodiments, the protein of interest can be associated with visible markers (including fluorescence), viability, fertility, fecundity, fitness, flight ability, vision, and behavioral differences. In some embodiments, the expression systems are conditional, with the one or more heterologous genes of interest being expressed only under some, for instance restrictive, conditions.

[0167] The term “heterologous” as used herein refers to a sequence that would not, in the wild type, be normally found in association with, or linked to, at least one element or component of the splice control module comprising the heterologous gene of interest at least one element or component of the gene expression polynucleotide comprising the heterologous gene of interest. For example, when the splice control module is derived from a particular organism, the heterologous gene of interest could be derived, in part or in whole, from a gene from the same organism, provided that in the organism’ s genome, the origin of at least some part of heterologous gene sequence (i.e., the sequence from which the heterologous gene is derived) is not found in association with, or linked to the sequence from which the splice control sequence is derived from. Alternatively, the heterologous gene could be from a different organism and, in this context, could be thought of as “exogenous”. The heterologous gene could also be considered as “recombinant,” in that the heterologous gene sequence is derived from different locations, either within the same genome (i.e., the genome of a single species or subspecies) or from different genomes (i.e., genomes from different species or subspecies), or synthetic sources.

[0168] One or more heterologous genes of interest can also be linked to a sequence other than the splice control module, such as a promoter and other sequences such as 5’ UTR and/or 3’UTR that is heterologous to the heterologous gene to be expressed in the organism, provided that said heterologous gene is not found in association or operably linked to the promoter, 5’ UTR and/or 3’UTR, in the wild type, i.e., the natural context of said polynucleotide sequence, if any.

[0169] It will be understood that heterologous also applies to “designer” or hybrid sequences that are not derived from a particular organism but are based on a number of components from different organisms. It will also be understood that synthetic versions of naturally occurring sequences are envisioned. This applies equally to where the heterologous polynucleotide is a polynucleotide for RNA interference.

[0170] In embodiments where the heterologous gene of interest to be expressed comprises a coding sequence for a protein or polypeptide (e.g., an effector protein coding sequence and/or a sex-specific lethal protein coding sequence), reference to expression in an organism refers to the provision of one or more transcribed RNA sequences, preferably mature mRNAs, but this may also refer to translated polypeptides in said organism.

[0171] In some embodiments, the heterologous gene of interest is an effector protein coding sequence, wherein the effector protein is lethal, deleterious, or sterilizing . In some embodiments, the system further comprises a second heterologous gene of interest that encodes a color marker. In some embodiments, at least one of the heterologous genes of interest is under the control of the splice control module as discussed in Sections II. A.1 and II. A.2.

[0172] In some embodiments, the effector protein is differentially expressed in different sexes due to differential splicing. In some embodiments, the effector protein is differentially expressed in different sexes not due to differential splicing. In some embodiments, the effector protein is naturally expressed in one sex but not in another sex. In some embodiments, the effector protein is lethal in a first sex when expressed in said first sex, but is not lethal in a second sex when expressed in said second sex. In some embodiments, the first sex is female and the second sex is male. [0173] In some embodiments, the effector protein is a sex-specific lethal protein that is lethal to one sex when expressed. For example, in some embodiments, the sex-specific lethal protein is YOB or GUY1, which is lethal to female Anopheles mosquitoes. In some embodiments, the system further comprises a second heterologous gene of interest that encodes a color marker. In some embodiments, at least one of the heterologous genes of interest is under the control of the gene expression modules as discussed in Section II.B.

[0174] In some embodiments, expression of the effector protein causes lethality in pre-adult females in the absence of a tetracycline analogue. In some embodiments, expression of the effector protein causes lethality in pre-adult females before they reach pupal stage in the absence of a tetracycline analogue. In some embodiments, expression of the effector protein causes lethality in pre-adult females, with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least >99.9% effectiveness. In some embodiments, expression of the effector protein causes lethality in pre-adult females, with at least 90% effectiveness. Effectiveness may be measured by, for example, the percentage of females that carry one of more copies of the lethal gene that are killed in pre-adult life stages..

[0175] Exemplary effector protein coding sequences include, but are not limited to, sequence coding for any of the following exemplary effector proteins: a tTA or a tTAV gene variant, or a variant thereof, GUY 1 or a variant thereof, ReaperKR or a variant thereof, YOB or a variant thereof, an apoptosis-inducing factor or a variant thereof, Hid or a variant thereof, and NipplDm or a variant thereof. In some embodiments, an effector protein coding sequence is differentially spliced, such as, for example, spliced in a sex- specific manner. For example, in some embodiments, a tTAV or a variant thereof coding sequence is spliced in a sex-specific manner. In some embodiments, an effector protein coding sequence is not differentially spliced between sexes. In some such embodiments, an effector protein is expressed in a sex-linked manner. In some embodiments, an effector protein is expressed in a sex-linked manner based on the coding and/or amino acid sequence of the effector protein. For example, in some embodiments, a GUY1 and/or a YOB effector protein or a variant thereof is sex- specifically expressed and active as a result of its sequence. In some embodiments, expression of an effector protein causes lethality in females but not in males

1. Lethal Genes

[0176] In some embodiments, the heterologous gene of interest (e.g., an effector protein coding sequence or a sex-specific lethal protein coding sequence) is a lethal gene that encodes a gene product (e.g., an effector protein) that may have lethal, deleterious, or sterilizing effects. Where reference is made herein to a lethal effect, it will be appreciated that this extends to a deleterious or sterilizing effect, such as an effect capable of killing the organism per se or its offspring, or capable of reducing or destroying the function of certain tissues such as the reproductive tissues, so that the organism or its offspring are sterile. Therefore, some lethal effects, such as poisons, will kill the organism or tissue in a short time-frame relative to their life-span, whilst others may simply reduce the organism’s ability to function, for instance reproductively.

[0177] The lethal protein may act on specific cells or tissues or impose its effect on the whole organism. The lethal protein may only have lethal effects in one specific sex, such as females, when expressed at similar levels in both sexes. Systems that are not strictly lethal but impose a substantial fitness cost are also envisioned, for example leading to blindness, flightlessness (for organisms that could normally fly), or sterility. Systems that interfere with sex determination are also envisioned, for example transforming or tending to transform all or part of an organism from one sexual type to another.

[0178] In some embodiments, the lethal effect may result in sterilization and/or may be produced in an organism that is otherwise sterile, for example, allowing the organism to compete in the natural environment (“in the wild”) with wild-type organisms, but without, in some embodiments, being able to produce viable offspring..

[0179] The present invention allows for selective control of the expression of the lethal gene, thereby providing selective control of the expression of a lethal phenotype. It will therefore be appreciated that each of the lethal genes encodes a functional protein, such as described in W02005/012534.

[0180] The lethal gene has a lethal effect that is conditional. An example of suitable conditions includes temperature, so that the lethal gene is expressed at one temperature but not, or to a lesser degree, at another temperature. Such conditional control could be achieved by using systems comprising GAL4. Another example of a suitable condition is the presence or absence of a substance, whereby the lethal gene is expressed in either the presence or absence of the substance, but not both. It is preferred that the effect of the lethal gene is conditional and is not expressed under permissive conditions requiring the presence of a substance (such as, for example, tetracycline and/or an analogue thereof) that is absent from the natural environment of the organism, such that the lethal effect of the lethal system occurs in the natural environment of the organism.

[0181] In other embodiments, the lethal genes is tTA or a tTAV gene variant, where tTA denotes ‘tetracycline repressible Trans-Activator’ and V denotes ‘Variant.’ tTAV is an analogue of tTA, wherein the sequence of tTA has been modified to enhance the compatibility with the desired insect species. Variants of tTAV are possible, encoding the tTA protein, such that the tTAV gene products have the same functionality as the tTA gene product. Thus, the variants of the tTAV gene comprise modified nucleotide sequences as compared to the tTA nucleotide sequence and to each other, but encode proteins with the same function. Thus, tTAV gene variants can be used in the place of tTA. In some embodiments the tTA Variant proteins contain amino acid substitutions, additions or deletions. Any combination of lethal genes may be used, and, in some embodiments, the lethal genes are the same, while in other embodiments, the lethal genes are different. The improved penetrance of the lethal effect and the earlier onset of lethality is achieved by an accumulation of lethal product.

[0182] In some embodiments, the system comprises at least one positive feedback mechanism. For example, the system may comprise a lethal protein to be differentially expressed via alternative splicing (by using a splice control module such as discussed in Section II. A.1 and Section II. A.2), and at least one promoter therefor, wherein the at least one lethal protein serves as a positive transcriptional control factor for the at least one promoter, and whereby the expression of the at least one lethal protein is controllable. In some embodiments, an enhancer is associated with the promoter, and lethal protein enhances the activity of the promoter via the enhancer. In a particular embodiment, the lethal gene linked to a splice control module is tTAV, and the splice control module is associated with neighboring tetO repeats. Upstream of a promoter, in either orientation, tetO is capable of enhancing levels of transcription from a promoter in close proximity thereto, when bound by the product of the tTA or tTAV gene. If the tTA or tTAV gene is part of the cassette comprising the tetO operator together with the promoter, then positive feedback occurs when the tTA gene product is expressed.

[0183] The tTA system also has the advantage of providing stage- specific toxicity. In particular, “squelching” is observed in the development phases of many insects, the precise phase of susceptible insects being species-dependent. Some insects may reach pupation before the larva dies, while others die early on. Susceptibility ranges from 100% fatality to a small reduction in survival rates. In general, though, adult insects appear to be immune to the squelching effect of tTA, so that it is possible to raise insects comprising a tTA positive feedback system in the presence of a tetracycline-class antibiotic, and then to release the adult insects into the wild. These insects are at little or no competitive disadvantage to the wild type, and will breed with the wild type insects, but larvae carrying the tTA positive feedback cassette will die before reaching maturity.

[0184] Tetracycline-class antibiotics, such as tetracycline and its analogues, include but are not limited to: tetracycline, chlortetracycline, oxy tetracycline, demeclocy cline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, doxycycline, tigecycline, eravacycline, sarecycline, and omadacycline.

[0185] In particular embodiments, the lethal gene is tTA or a tTAV gene variant. In some embodiments, the lethal gene is tTAV (SEQ ID NO: 18) and the lethal protein is tTAV comprising the sequence set forth in SEQ ID NO: 19. In some embodiments, the lethal gene is tTAV2 (SEQ ID NO: 20) and the lethal protein is tTAV2 comprising the sequence set forth in SEQ ID NO: 21. In some embodiments, the lethal gene is tTAV3 (SEQ ID NO: 22) and the lethal protein is tTAV3 comprising the sequence set forth in SEQ ID NO: 23. In some embodiments, tTA or the tTAV gene variant is differentially expressed in a sex-specific manner due to sex-specific alternative splicing.

[0186] NipplDm, the Drosophila homologue of mammalian nuclear inhibitor of PPI (Nippl) (Parker et al. (2002) Biochemical Journal 368:789-797; Bennett et al., (2003) Genetics 164:235-245) is utilized in some embodiments. NipplDm is another example of a protein with lethal effect if expressed at a suitable level, as would be understood by the skilled person. In some embodiments, the lethal gene is NipplDm.

[0187] In some embodiments, the product of at least one of the lethal genes is an apoptosisinducing factor, such as the AIF protein described for instance in Cande et al. (2002) (J. Cell Science 115:4727-4734) or homologues thereof. Apoptosis-inducing factor (AIF) is a mitochondrial oxidoreductase that contributes to cell death programs and participates in the assembly of the respiratory chain. In some embodiments, the lethal gene is AIF.

[0188] In some embodiments, the lethal gene is Hid. The activation of Hid expression could cause lethality due to induction of apoptosis (Bilak and Su, (2009), Apoptosis 14, 943-949.). Use of Hid was described by Heinrich and Scott (2000) Proc. Natl Acad. Sci USA 97:8229- 8232). Use of a mutant derivative, HidAla5 was described by Hom and Wimmer (2003) Nature Biotechnology 21:64-70).

[0189] In other embodiments, the lethal gene is Reaper (Rpr) (SEQ ID NO: 26) and the lethal protein comprises a sequence set forth in SEQ ID NO: 27. Use of a mutant derivative of Rpr, RprKR (SEQ ID NO: 30), is described herein (see also White et al. (1996), Science 271(5250):805-807; Wing et al. (2001) Meeh. Dev. 102(1-2): 193-203; and Olson et al. (2003) J. Biol. Chem. 278(45):44758-44768). Both Rpr and Hid are pro-apoptotic proteins, thought to bind to IAP1. IAP1 is a well-conserved anti-apoptotic protein. Hid and Rpr are therefore expected to work across a wide phylogenetic range (Huang et al. (2002); Vemooy et al. (2000) J. Cell Biol. 150(2):F69-76) even though their own sequence is not well conserved.

[0190] In some embodiments, the lethal gene is a sequence coding for a sex-specific lethal protein, such as a female- specific lethal protein. In some embodiments, the female-specific lethal protein is GUY1. The GUY1 protein is a primary signal from the Y chromosome that affects embryonic development in a sex-specific manner (Criscione et al., (2016) eLife 2016; 5: el9281). The GUY1 gene is identified only on the Y chromosome (Criscione et al., 2013). Female An. stephensi mosquitoes die when the GUY1 gene is placed on and expressed from their non-sex chromosomes. Without wishing to be bound by any theory, GUY1 maybe be involved in the regulation of dosage compensation in An. stephensi, and ectopic expression of GUY1 in XX individuals may result in higher-than-normal levels of expression from X-linked genes, which could be lethal. In some embodiments, the coding sequence of GUY1 comprises the sequence set forth in SEQ ID NO: 28. In some embodiments, the GUY 1 protein comprises the sequence set forth in SEQ ID NO: 29. In some embodiments, GUY 1 exhibits lethal effects in a sex-specific manner in the absence of differential sex-specific splicing.

[0191] In some embodiments, the female- specific lethal protein is YOB, encoded by the gene Yob, a Y chromosome-linked gene that may also be involved in the regulation of dosage compensation. In Anopheles gambiae, silencing embryonic Yob expression is male-lethal, whereas ectopic embryonic delivery of Yob transcripts is lethal for genetically female embryos (Krzywinska et al., (2016) Science 353(6294):67-9; Krzywinska & Krzywinski, 2018). In some embodiments, the coding sequence of YOB comprises the sequence set forth in SEQ ID NO:

31. In some embodiments, the YOB protein comprises the sequence set forth in SEQ ID NO:

32. In some embodiments, YOB exhibits lethal effects in a sex-specific manner in the absence of differential sex-specific splicing. 2. RNAi

[0192] The heterologous gene of interest to be expressed may comprise polynucleotides for RNA interference (RNAi). In some embodiments, where the heterologous gene of interest to be expressed comprises polynucleotides for RNAi, it will also be understood that reference to expression in an organism refers to the interaction of the polynucleotides for RNAi, or transcripts thereof, in the RNAi pathway, for instance by binding of Dicer (RNA Pol Ill-like enzyme) or formation of small interfering RNA (siRNA). Such sequences are capable of providing, for instance, one or more stretches of double- stranded RNA (dsRNA), preferably in the form of a primary transcript, which in turn is capable of processing by the Dicer. Such stretches include, for instance, stretches of single- stranded RNA that can form loops, such as those found in short-hairpin RNA (shRNA), or with longer regions that are substantially self- complementary. Antisense sequences or sequences having homology to microRNAs that are naturally occurring RNA molecules targeting protein 3’ UTRs are also envisaged as sequences for RNAi.

[0193] It will also be understood that where the system is DNA, the polynucleotides for RNA interference are deoxyribonucleotides that provide a stretch of dsRNA when transcribed into pre-RNA ribonucleotides.

[0194] In some embodiments, polynucleotides for RNA interference are positioned to minimize interference with alternative splicing. This may be achieved by distal positioning of these polynucleotides from the alternative splice control sequences, preferably 3’ to the control sequences. In another embodiment, substantially self-complementary regions may be separated from each other by one or more splice control sequences, such as an intron, that mediate alternative splicing. Preferably, the self-complementary regions are arranged as a series of two or more inverted repeats, each inverted repeat separated by splice control sequence, preferably an intron, as defined elsewhere.

[0195] In this configuration, different alternatively spliced transcripts may have their substantially self-complementary regions separated by different lengths of non-self- complementary sequence in the mature (post- alternative- splicing) transcript. It will be appreciated that regions that are substantially self-complementary are those that are capable of forming hairpins, for instance, as portions of the sequence are capable of base-pairing with other portions of the sequence. These two portions do not have to be exactly complementary to each other, as there can be some mismatching or toleration of stretches in each portion that do not base-pair with each other. Such stretches may not have an equivalent in the other portion, such that symmetry is lost and “bulges” form, as is known with base-pair complementation in general.

[0196] In another embodiment, one or more segment of sequence substantially complementary to another section of the primary transcript is positioned, relative to the at least one splice control sequence, so that it is not included in all of the transcripts produced by alternative splicing of the primary transcript. By this method, some transcripts are produced that tend to produce dsRNA while others do not; by mediation of the alternative splicing, e.g., sex-specific mediation, stage-specific mediation, germline- specific mediation, tissue- specific mediation, and combinations thereof, dsRNA may be produced in a sex-specific, stage- specific, germline- specific or tissue-specific manner, or combinations thereof.

3. Promoters, 5 ’ UTRs, and. 3 ’ UTRs

[0197] In one aspect, provided herein is a gene expression system comprising the splice control module polynucleotides disclosed herein (such as a dsx splice control module polynucleotide as described in Sections II. A.1 and II.A.2.a). In some embodiments, the gene expression system further comprises a 5’ untranslated region (5’ UTR) operably linked 5’ of the splice control module polynucleotide. In some embodiments, the gene expression system further comprises a promoter operable in an insect. The promoter is capable of being activated by an activating transcription factor or trans-activating factor. It is understood that any combination of promoter and splice control module can be envisaged. The promoter is preferably specific to a particular protein having a short temporal or confined spatial effect, for example a cell-autonomous effect.

[0198] The promoter may be a large or complex promoter, but these often suffer the disadvantage of being poorly or patchily utilized when introduced into non-host insects. Accordingly, in some embodiments, it is preferred to employ minimal promoters. It will be appreciated that minimal promoters may be obtained directly from known sources of promoters, or derived from larger naturally occurring, or otherwise known, promoters. Suitable minimal promoters and how to obtain them will be readily apparent to those skilled in the art. For example, suitable minimal promoters include a minimal promoter derived from Hsp70, a P minimal promoter, a CMV minimal promoter, an Act5C-based minimal promoter, a BmA3 promoter fragment, and an Adh core promoter (Bieschke, E. et al. (1998) Mol. Gen. Genet., 258:571-579). Not all minimal promoters will necessarily work in all species of insect, but it is readily apparent to those skilled in the art as to how to ensure that the promoter is active. It is preferred that at least one of the operably-linked promoters present in the invention is active during early development of the host organism, and particularly preferably during embryonic stages, in order to ensure that the lethal gene is expressed during early development of the organism.

[0199] In some embodiments, the promoter can be activated by environmental conditions, for instance the presence or absence of a particular factor such as tetracycline in the tet system described herein, such that the expression of the gene of interest can be easily manipulated by the skilled person. Alternatively, a preferred example of a suitable promoter is the hsp70 heat shock promoter, allowing the user to control expression by variation of the environmental temperature to which the hosts are exposed in a lab or in the field, for instance. Another preferred example of temperature control is described in Fryxell and Miller (1995) J. Econ. Entomol. 88:1221-1232.

[0200] The promoter may be specific for a broader class of proteins or a specific protein that has a long-term and/or wide system effect, such as a hormone, positive or negative growth factor, morphogen or other secreted or cell-surface signaling molecule. This would allow, for instance, a broader expression pattern so that a combination of a morphogen promoter with a stage- specific alternative splicing mechanism could result in the morphogen being expressed only once a certain life-cycle stage was reached, but the effect of the morphogen would still be felt (i.e., the morphogen can still act and have an effect) beyond that life-cycle stage. Such examples include the morphogen/signaling molecules Hedgehog, Wingless/WNTs, TGFB/BMPs, EGF and their homologues, which are well-known evolutionarily-conserved signaling molecules.

[0201] It is also envisaged that a promoter that is activated by a range of protein factors, for instance transactivators, or which has a broad systemic effect, such as a hormone or morphogen, could be used in combination with an alternative splicing mechanism to achieve a tissue and sex-specific control or sex and stage-specific control, or other combinations of stage- , tissue-, germ-line- and sex-specific control.

[0202] It is also envisaged that more than one promoter, and optionally an enhancer therefor, can be used in the present system, either as alternative means for initiating transcription of the same protein or by virtue of the fact that the genetic system comprises more than one gene expression system (i.e., more than one gene and its accompanying promoter).

[0203] In some embodiments, the promoter operably linked to the splice control module is selected from the group consisting of: Drosophila melanogaster minimal HSP70 promoter (DmHsp70), TRE3G, CMV minipromoter, OpIE2, Vasa, bZIPl, and Act5c (SEQ ID NOs: 35- 44). When an activating transcription factor activates the promoter, the expression of the lethal gene operably linked to the promoter is up-regulated. The activating transcription factor may act in any suitable manner. For example, the activating transcription factors may bind to an enhancer located in proximity to the at least one promoter, thereby serving to enhance polymerase binding at the promoter. Other mechanisms may be employed, such as repressor countering mechanisms, such as the blocking of an inhibitor of transcription or translation. Transcription inhibitors may be blocked, for example, by the use of hairpin RNA’s or ribozymes to block translation of the mRNA encoding the inhibitor, or the product may bind the inhibitor directly, thereby preventing inhibition of transcription or translation.

[0204] The splice control modules described herein further comprises a 3’ UTR sequence. In some embodiments, the 3’ UTR comprises a 3’ sequence of exon 6. In some embodiments, the 3’ UTR sequence is derived from a PIO 3’ UTR or a SV40 3’ UTR. PIO 3’ UTR and SV40 3’ UTR contribute to the efficient termination of transcription and polyadenylation, and can increase the expression of the heterologous sequence of interest (e.g., an effector protein such as a lethal protein). In some embodiments, the 3’ UTR sequence is derived from PIO 3’ UTR comprising the sequence set forth in SEQ ID NO: 33. In some embodiments, the 3’ UTR sequence is derived from SV40 3’ UTR comprising the sequence set forth in SEQ ID NO: 34. In some embodiments, the 3’ UTR sequence is derived from YOB 3’ UTR comprising the sequence set forth in SEQ ID NO: 68. In some embodiments, the 3’ UTR sequence is derived from GUY1 3’ UTR comprising the sequence set forth in SEQ ID NO: 69.

4. Repressible Elements

[0205] Preferably, the gene expression system comprising the lethal gene as discussed in Section II.C.l is a dominant lethal genetic system, the lethal effect of which is conditional. Suitable conditions include, for example, temperature (so that, for example, the lethal gene is expressed at one temperature but not, or to a lesser degree, at another temperature) and presence or absence of a compound (such as, for example tetracycline or an analogue thereof). The lethal gene product may act on specific cells or tissues or impose its effect on the whole organism. It will be understood that all such systems and consequences are encompassed by the term lethal as used herein. Similarly, “killing”, and similar terms refer to the effective expression of the lethal gene and thereby the imposition of a deleterious or sex-distorting phenotype, such as death. In some embodiments of the present invention, the lethal effect is specific to a particular sex (e.g., female).

[0206] More preferably, the gene expression system is a recombinant dominant lethal genetic system, the lethal effect of which is conditional and is not expressed under permissive conditions requiring the presence of a substance which is absent from the natural environment of the organism, such that the lethal effect of the system occurs in the natural environment of the organism.

[0207] In some embodiments, the lethal gene (such as an effector protein coding sequence described in Section II. A.1 or a sequence coding for a female-specific lethal protein described in Section II.B) is linked to a system such as the tet system described in WO 01/39599 and W02005/012534.

[0208] Indeed it is preferred that the expression of said lethal gene is under the control of a repressible transactivator protein. It is also preferred that the gene whose expression is regulated by alternative splicing encodes a transactivator protein such as tTA or tTAV. This is not incompatible with the regulated protein being a lethal. Indeed, it is particularly preferred that it is both. In this regard, we particularly prefer that the system includes a positive feedback system as taught in W02005/012534 and discussed in Section II.C.l.

[0209] In some embodiments, the lethal gene is tTA or a tTAV gene variant, and an enhancer is a tetO element, comprising one or more tetO operator units (repeats). An example of a tetO operator unit is set forth in SEQ ID NO: 57. Upstream of a promoter, in either orientation, tetO is capable of enhancing levels of transcription from a promoter in close proximity thereto, when bound by the product of the tTA gene or a tTAV gene variant. In some embodiments, the enhancer is selected from the group consisting of tetOxl, tetOx2, tetOx3, tetOx4, tetOx5, tetOx6, tetOx7, tetOx8, tetOx9, tetOxlO, tetOxl 1, tetOxl2, tetOxl3, tetOxl4, tetOxl5, tetOxl6, tetOxl7, tetOxl8, tetOxl9, tetOx20, and tetOx21. In some embodiments, the enhancer is tetOxl, tetOx9, tetOxl5, tetOxl7, or tetOx21. An example of the TetOx7 element is shown in SEQ ID NO: 58. An example of the TetOx21 element is shown in SEQ ID NO: 59. 5. Fusion Leaders

[0210] In some embodiments such as those shown in FIGs. 1B-1C, it will be desirable to have the heterologous protein of interest linked to the splice control module free of the splice control module protein sequence. In some embodiments, the splice control module is operatively linked to a polypeptide-encoding polynucleotide that stimulates proteolytic cleave of a translated polypeptide (“Fusion Leader Sequence” for the polynucleotide and “Fusion Leader Polypeptide” for the encoded polypeptide). An example of such a Fusion Leader Sequence is ubiquitin encoding polynucleotide. Such a Fusion Leader Sequence may be operatively linked in frame to the 3’ end of the splice control module polynucleotide and operatively linked in frame to the gene of interest (i.e., from 5’ to 3’: Splice Control Module- Fusion Leader Sequence-Gene of interest). In such a case, the Splice Control Module/Fusion Leader Polypeptide is cleaved from the protein of interest by specific proteases in the cell. Aside from ubiquitin, any other similar fusion may be made in place of ubiquitin that would have the effect of stimulating a cleavage of the N-terminal Splice Control Module. In some embodiments, the Fusion Leader Polypeptide is ubiquitin comprising the sequence set forth in SEQ ID NO: 61.

[0211] Therefore in some embodiments, provided herein is a gene expression system comprising from 5’ to 3’: i) a doublesex (dsx') splice control module polynucleotide operably linked to an effector protein coding sequence wherein the splice control module comprises (from 5’ to 3’): a dsx exon 4 sequence, a dsx intron 4 sequence, a dsx exon 5 sequence, a dsx intron 5 sequence, and a dsx exon 6 sequence; ii) a Fusion Leader Sequence; and iii) effector protein coding sequence (such as a lethal gene). In some embodiments, the Fusion Leader Sequence encodes a Fusion Leader Polypeptide such as ubiquitin comprising the sequence set forth in SEQ ID NO: 61.

6. Other elements

[0212] In some embodiments, the system comprises other upstream, 5’ factors and/or downstream 3’ factors for controlling expression. Examples include enhancers such as the fatbody enhancers from the Drosophila yolk protein genes, and the homology region (hr) enhancers from baculoviruses, for example AcNPV Hr5.

[0213] It will be understood that reference is made to start and stop codons between which the polynucleotide sequence to be expressed in an organism is defined, but that this does not exclude positioning of the splice control module polynucleotide, elements thereof, or other sequences, such as introns, in this region. In fact, it will be apparent form the present description that the splice control sequence, can, in some embodiments, be positioned in this region.

[0214] Furthermore, the splice control sequence, for instance, can overlap with the start codon at least, in the sense that the G of the ATG can be, in some embodiments, be the initial 5’ G of the splice control sequence. Thus, the term “between” can be thought of as referring to from the beginning (3’ to the initial nucleotide, i.e., A) of the start codon, preferably 3’ to the second nucleotide of the start codon (i.e., T), up to the 5’ side of the first nucleotide of the stop codon. Alternatively, as will be apparent by a simple reading of a polynucleotide sequence, the stop codon may also be included.

D. Vectors

[0215] One aspect of the present invention relates to an expression vector plasmid comprising the gene expression system or the gene expression polynucleotide disclosed herein. The expression vector plasmid can be either DNA, RNA or a mixture of both. If the expression vector plasmid comprises RNA, then it may be preferable to reverse-translate the RNA into DNA by means of a Reverse Transcriptase (RTase). If reverse transcription is required, then the expression vector plasmid may also comprise a coding sequence for the RTase protein and a suitable promoter therefor. Alternatively, the RTase and promoter therefore may be provided on a separate system, such as a virus. In this case, the system would only be activated following infection with that virus. The need to include suitable cis-acting sequences for the reverse transcriptase or RNA-dependent RNA polymerase would be apparent to the person skilled in the art.

[0216] However, it is particularly preferred that the system is predominantly DNA and more preferably consists only of DNA, at least with respect to the sequences to be expressed in the organism.

[0217] In some embodiments, the expression vector plasmid further comprises a polynucleotide encoding a color marker protein. In some embodiments, the color marker protein is a fluorescent marker protein. In some embodiments, fluorescent marker protein is GFP, ZsGreenl, mCherry, DsRed-Monomer, DsRed-Express, DsRed-Express2, tdTomato, AsRed2, mStrawberry, E2-Crimson, HcRedl, mRaspberry, mPlum, mOrange2, mBanana, ZsYellowl, Dendra2, PAmCherry, Timer, DsRed2, or AmCyanl. In some embodiments, the polynucleotide encoding DsRed2 comprises the sequence set forth in SEQ ID NO: 54. In some embodiments, the polynucleotide encoding AmCyanl comprises the sequence set forth in SEQ ID NO: 53.

[0218] In some embodiments, the polynucleotide encoding the color marker protein (such as the fluorescent marker protein) is operably linked to a promoter operable in an insect. In some embodiments, the promoter is an IE1 promoter or a 3xP3 promoter. In some embodiments, the IEI promoter comprises a sequence set forth in SEQ ID NO: 47. In some embodiments, the 3xP3 promoter comprises a sequence set forth in SEQ ID NO: 48.

[0219] In some embodiments, the expression vector plasmid further comprises an enhancer to enhance expression. In some embodiments, the enhancer is Hr5 enhancer, a non-coding fragment from Autographa californica nucleopolyhedrovirus (AcNPV). In some embodiments, the Hr5 enhancer is truncated. For example an Hr5 enhancer can be truncated by reducing the number of CRE repeats to tune down gene expression. An example of a truncated Hr5 enhancer is set forth in SEQ ID NO: 46.

[0220] In some embodiments, the expression vector plasmid of the present invention comprises the splice control module disclosed in Section II.A or the gene expression polynucleotide disclosed in Section II.B.

[0221] In some embodiments, the expression vector plasmid comprises a sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 1-17. In some embodiments, the expression vector plasmid comprises a sequence set forth in any one of SEQ ID NOs: 1-17.

E. Introduction of Constructs into Organisms

[0222] Methods of introduction or transformation of the gene system constructs and induction of expression are well known in the art with respect to the relevant organism. It will be appreciated that the system or construct is preferably administered as a plasmid, but generally tested after integrating into the genome. Administration can be by known methods in the art, such as parenterally, intra- venously, intra-muscularly, orally, transdermally, delivered across a mucous membrane, and so forth. Injection into embryos is particularly preferred. The plasmid may be linearized before or during administration, and not all of the plasmid may be integrated into the genome. Where only part of the plasmid is integrated into the genome, it is preferred that this part include the at least one splice control module capable of mediating alternative splicing.

[0223] Plasmid vectors may be introduced into the desired host cells by methods known in the art, such as, for example by transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., (1992) J. Biol. Chem. 267:963; Wu et al. (1988) J. Biol. Chem. 263:14621; and Canadian Patent Application No. 2,012,311 to Hartmut et al.). The plasmid vector may be integrated into the host chromosome by any means known. Well-known methods of locus- specific insertion may be used, including, homologous recombination and recombinase-mediated genome insertion. In another embodiment, locusspecific insertion may be carried out by recombinase-site specific gene insertion. In one example piggyBac sequences (such as SEQ ID NOs: 45, and 63-64) may be incorporated into the vector to drive insertion of the vector into the host genome. In some embodiments, Mariner transposable elements can be used to introduce the vector into the host genome. Other technologies such as CRISPRs, TALENs, PhiC31, ZFNs, AttP/AttB recombination may also be employed.

F. Genetically Engineered Organisms

[0224] In one aspect, there is provided a genetically engineered insect comprising a gene expression system incorporated into a chromosome of said insect, said gene expression system comprising a polynucleotide construct comprising: i. a doublesex (dsx) splice control module wherein said splice control module comprises the components from 5’ to 3’:i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a first portion of a dsx exon 5 sequence comprising a 5’ terminal fragment of dsx exon 5; iv) an effector protein coding sequence; v) a second portion of a dsx exon 5 sequence comprising a 3’ terminal fragment of dsx exon 5; vi) a dsx intron 5; and vii) a dsx exon 6; and ii. a 5’ UTR positioned 5’ of said splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect. In some embodiments, the dsx splice control module is derived from an Anopheles dsx.

[0225] In one aspect, there is provided a genetically engineered insect comprising a gene expression system incorporated into a chromosome of said insect, said gene expression system comprising: i. a doublesex (dsx) splice control module polynucleotide operably linked to an effector protein coding sequence, wherein the dsx splice control module comprises, from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a dsx exon 5 sequence; iv) a dsx intron 5 sequence; and v) a dsx exon 6 sequence; wherein the effector protein coding sequence is 3’ of the dsx splice control module polynucleotide; ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the effector protein coding sequence; and iv. a promoter operable in the insect.

[0226] In another aspect, there is provided a female genetically engineered insect comprising a gene expression system incorporated into a chromosome of said insect, said gene expression system comprises: i. a doublesex (dsx) splice control module wherein said splice control module is capable to produce a splice form comprising the components from 5’ to 3’: i) a dsx exon 4 sequence; iii) a first portion of a dsx exon 5 sequence comprising a 5’ terminal fragment of dsx exon 5; iv) an effector protein coding sequence, wherein the effector protein is lethal, deleterious sterilizing, or otherwise leading to a dosage sex determination bias to an insect; v) a second portion of a dsx exon 5 sequence comprising a 3’ terminal fragment of dsx exon 5; vii) a dsx exon 6, wherein the dsx splice control module is spliced specifically in the female genetically engineered insect to produce one or more female splice forms that express the effector protein;; and ii. a 5’ UTR positioned 5’ of said splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect.

[0227] In another aspect, there is provided a female genetically engineered insect comprising a gene expression system incorporated into a chromosome of said insect, said gene expression system comprises: a first module comprising from 5’ to 3’: i. a promoter operable in an insect; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR; and a second module comprising from 5’ to 3’ : i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a sex-specific lethal protein; and iv. a 3’ UTR. In some embodiments, the sex-specific lethal protein is a female- specific lethal protein. In some embodiments, the female- specific lethal protein is GUY1 or a variant thereof. In some embodiments, the female-specific lethal protein is YOB or a variant thereof.

[0228] In another aspect, there is provided a method of producing a genetically engineered insect comprising inserting a gene expression system into the insect’s genome, wherein said gene expression system comprises: i. a doublesex (dsx) splice control module wherein said splice control module comprises the components from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a first portion of a dsx exon 5 sequence; iv) an effector protein coding sequence; v) a second portion of a dsx exon 5 sequence; vi) a dsx intron 5; and vii) a dsx exon 6; and ii. a 5’ UTR positioned 5’ of said splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect.

[0229] In another aspect, there is provided a method of producing a genetically engineered insect comprising inserting a gene expression system into the insect’s genome, wherein said gene expression system comprises: i. a doublesex (dsx') splice control module polynucleotide operably linked to an effector protein coding sequence, wherein the dsx splice control module comprises, from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a dsx exon 5 sequence; iv) a dsx intron 5 sequence; and v) a dsx exon 6 sequence; wherein the effector protein coding sequence is 3’ of the dsx splice control module polynucleotide; ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the effector protein coding sequence; and iv. a promoter operable in the insect.

[0230] In another aspect, there is provided a method of producing a genetically engineered insect comprising inserting a gene expression system into the insect’s genome, wherein said gene expression system comprises: a first module comprising from 5’ to 3’: i. a promoter operable in an insect; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR; and a second module comprising from 5’ to 3’: i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a sex-specific lethal protein; and iv. a 3’ UTR. In some embodiments, the sex-specific lethal protein is a female- specific lethal protein. In some embodiments, the female- specific lethal protein is GUY1 or a variant thereof. In some embodiments, the female-specific lethal protein is YOB or a variant thereof.

[0231] The vectors of the invention may be used to create genetically engineered organisms in a wide variety of genera and species. In some embodiments, the insect is of the Order Diptera.

[0232] In some embodiments, the insect is a mosquito of a genus selected from the group consisting of Anopheles, Stegomyia, Aedes, and Culex. Examples include Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles spp., and Anopheles gambiae.

[0233] Other Diptera species suitable for transformation with a vector of the invention include, but are not limited to Calliphoridae species selected from the group consisting of New world screwworm (Cochliomyia hominivorax), Old world screwworm (Chrysomya bezziana), and Lucilia cuprinay tephritid fruit flies such as Medfly (Ceratitis capitata), Mexfly (Anastrepha ludens). Oriental fruit fly (Bactrocera dorsalis), Olive fruit fly (Bactrocera oleae), Melon fly (Bactrocera cuciirbilae), Natal fruit fly (Ceratitis rosa), Cherry fruit fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyroni), Peach fruit fly (Bactrocera zonatd), Caribbean fruit fly (Anastrepha suspensa). and West Indian fruit fly (Anastrepha obliqua).

[0234] In one aspect, there is provided a method of producing a genetically engineered insect comprising inserting a gene expression system into the insect’s genome, wherein said gene expression system comprises: i. a doublesex (dsx) splice control module wherein said splice control module comprises the components from 5’ to 3’: i). a dsx exon 4 sequence; ii). a dsx intron 4 sequence; iii). a first portion of a dsx exon 5 sequence; iv). an effector protein coding sequence; v). a second portion of a dsx exon 5 sequence; vi). a dsx intron 5; vii). a dsx exon 6; ii. a 5’ UTR positioned 5’ of said splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect.

[0235] In some embodiments, the dsx splice control module is derived from an Anopheles dsx such as those descried in Section II. A.1 and Section II.A.2.a. In some embodiments, the genetically engineered insect is a mosquito. The mosquito could be of the genus Anopheles, Aedes, Stegomyia or Culex. For example, the mosquito could be Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles spp., or Anopheles gambiae.

[0236] In one aspect, there is provided a method of producing a genetically engineered insect comprising inserting a gene expression system into the insect’s genome, wherein said gene expression system comprises: a first module comprising from 5’ to 3’: i. a promoter operable in an insect; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR; and a second module comprising from 5’ to 3’: i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a sex-specific lethal protein; and iv. a 3’ UTR. In some embodiments, the sex-specific lethal protein is a female- specific lethal protein. In some embodiments, the female- specific lethal protein is GUY1 or a variant thereof. In some embodiments, the female-specific lethal protein is YOB or a variant thereof.

[0237] In some embodiments, the gene expression system further comprises a polynucleotide encoding a fluorescent protein, as described in Section II.C.

G. Methods of Biological Control

[0238] In another aspect, there is provided a method of selectively rearing male genetically engineered insects comprising rearing a genetically engineered insect as discussed in Section ILF, wherein said rearing is in the absence of a tetracycline analogue.

[0239] In another aspect, there is provided a male genetically engineered insect produced by the method of selectively rearing male genetically engineered insects disclosed herein.

[0240] In a further aspect, there is also provided a method of reducing a wild insect population comprising contacting said wild insect population with a plurality of the male genetically engineered insects disclosed herein wherein said male genetically engineered insects mate with wild female insects.

[0241] Therefore in some embodiments, there is provided a method of reducing a wild insect population comprising contacting said wild insect population with a plurality of the male genetically engineered insects disclosed herein wherein said male genetically engineered insects mate with wild female insects, wherein the wild insect population is of the same species as the male genetically engineered insects disclosed herein. In some such embodiments, the male genetically engineered insects pass on a conditionally expressed lethal gene to offspring produced with the wild female insects, and the lethal gene is expressed in at least a portion of such offspring (e.g., in at least a portion of the female offspring), producing deleterious or lethal effects in at least a portion of the offspring (e.g., females).

[0242] In some embodiments, male genetically engineered insects mate with wild female insects of the same species.

[0243] In some embodiments, male genetically engineered insects mate with wild female insects of a different species.

[0244] Preferably, the lethal effect of the lethal system is conditional and occurs in the said natural environment via the expression of a lethal gene, the expression of the lethal gene being under the control of a repressible transactivator protein, the said breeding being under permissive conditions in the presence of a substance, the substance being absent from the natural environment and able to repress said transactivator. In some embodiments, the lethal trait is sufficiently repressible in female insects in the presence of a tetracycline analogue. For example, in some embodiments, in the presence of a tetracycline analogue, females can be reared healthily and can lay fertile eggs, to enable production colonies to be maintained.

[0245] In some embodiments, the system also provides high female lethality. In some embodiments, the lethal gene is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% effective in killing females in pre-adult life stages in the absence of the tetracycline analogue. In some embodiments, the lethal gene is at least about 99% effective in killing females in pre-adult life stages in the absence of the tetracycline analogue. The effectiveness is measured as the percentage of females that carry one of more copies of the lethal gene that are killed in pre-adult life stages.

[0246] In some embodiments, the system also provides male tolerance of the lethal effect. For example, in some embodiments, the effector gene is expressed in a sufficiently female sexspecific manner that it does not significantly kill or impair male fitness in the absence of the tetracycline analogue.

[0247] Also provided is a method of biological control, comprising: i) breeding a stock of males and female organisms transformed with the expression system according to the present invention under permissive conditions, allowing the survival of males and females, to give a dual sex biological control agent; ii) optionally before the next step imposing or permitting restrictive conditions (e.g., removing tetracycline analogues from the environments) to cause death of individuals of one sex (e.g., females) and thereby providing a single sex biological control agent comprising individuals of the other sex (e.g., males) carrying the conditional lethal genetic system; iii) releasing the dual sex or single sex biological control agent into the environment at a locus for biological control; and iv) achieving biological control through expression of the genetic system in offspring resulting from interbreeding of the individuals of the biological control agent with individuals of the opposite sex of the wild population.

[0248] The invention also provides methods of suppressing populations of wild insects, such as mosquitoes, comprising releasing genetically engineered male insects disclosed herein, among a population of wild insects of the same species, whereupon the genetically engineered insects mate with the wild insects and the female offspring die in pre-adult life stages thereby suppressing the population of wild insects.

[0249] The invention also provides methods of resistance management comprising releasing genetically engineered male insects disclosed herein, among a population of wild insects of the same species, wherein the population contains a plurality of insects that are resistant to insecticides, whereupon the genetically engineered insects mate with the wild insects and the female offspring die in pre-adult life stages. Surviving male offspring from such matings with wild females effectively also pass on the lethal gene disclosed herein (i.e., the traits are introgressed into the wild population), and dilute the frequency of resistance in the wild pest population. Further description of such a strategy may be found, for example in W02004098278. In this way, the method thereby suppresses the population of wild insects and slows or reverses resistance to insecticides in the population of wild insects.

[0250] Preferably, there is sex-separation prior to organism distribution by expression of a sex specific lethal genetic system.

[0251] Preferably, the lethal effect results in killing of greater than 90% of the target class of the progeny of matings between released organisms and the wild population, such as, for example, killing 90% or more of female progeny of matings between released organisms and the wild population.

[0252] Also provided is a method of sex separation comprising: i) breeding a stock of male and female organisms transformed with the gene expression system under permissive or restrictive conditions, allowing the survival of males and females; and ii) removing the permissive or restrictive conditions to induce the lethal effect of the lethal gene in one sex and not the other by sex-specific alternative splicing. In some embodiments, the method of sex separation comprises i) breeding a stock of male and female organisms transformed with the gene expression system in the presence of the tetracycline analogue, allowing the survival of males and females; and ii) removing the tetracycline analogue to induce the lethal effect of the lethal gene in females and not males by sex-specific alternative splicing.

[0253] Preferably, the lethal effect results in killing of greater than 90% of the target class of the progeny of matings between released organisms and the wild population, such as, for example, killing 90% or more of female progeny of matings between released organisms and the wild population.

[0254] Also provided is a method to selectively eliminate females from a population. The equivalent for males is also envisaged.

[0255] The invention will now be described by reference to the following examples which are meant to be illustrative of embodiments of the invention and are not to be construed as limiting the invention.

EXAMPLES

Example 1 - Genetically Engineered Anopheles via Splice Control

[0256] Genetically engineered Anopheles stephensi and An. albimanus strains were generated by insertion of one of the recombinant DNA (rDNA) constructs generated into the An. stephensi or An. Albimanus genome. The DNA constructs generally comprise two gene cassettes between the 5’ and 3’ fragments of the Trichoplusia ni piggyBac transposase used to insert them into the insect genome: (1) a splice control cassette that is linked to an effector protein (such as tTAV) encoding DNA by one of the three architectures discussed below, and (2) a color marker cassette for expressing either AmCyanl or DsRed2. The components of the DNA constructs are listed in Table El A.

[0257] Together these gene cassettes deliver An. stephensi and An. albimanus strains that, when reared in the presence of a tetracycline analogue (e.g., tetracycline-class antibody), development occurs normally in both sexes, but when reared in the absence of tetracycline females do not survive to adulthood and a male-only cohort is produced. Additionally, each insect is marked with the fluorescent AmCyanl or DsRed2 protein. Table E1A. Components of transgenes.

A. Sex-specific expression of the effector protein

[0258] Expression of the effector protein (such as tTAV) is rendered female- specific by the splice control module that is derived from either An. stephensi doublesex ( AstcD.sx) or An. albimanus doublesex (AalbD.sx). Three architectures were designed to achieve such femalespecific expression via dsx:

(a) Architecture 1: effector protein encoding DNA is incorporated into dsx exon 5, which is female- specific (FIG. 1A);

(b) Architecture 2: effector protein encoding DNA is downstream of the dsx splicing control module, and the translation of the dsx splicing control module starts from exon 4 (FIG.

IB); and

(c) Architecture 3: effector protein encoding DNA is downstream of the dsx splicing control module, and the translation of the dsx splicing control module starts from exon 5 (FIG.

IC).

[0259] In Architecture 1, the effector protein (such as tTAV) as part of exon 5, is retained in transcripts in females and spliced out in transcripts in males, and expressed as a standalone protein rather than part of a fusion protein as it has its own start codon and stop codon (FIG. 1A). [0260] In Architecture 2 and Architecture 3, the female splice form has a continuous open reading frame extending from either exon 4 (or a portion thereof) or exon 5 to the end of the effector protein, whereas the male splice form comprises a stop codon in exon 6 thus preventing the effector protein to be expressed. Architecture 2 and Architecture 3 also comprise a Leading Peptide (e.g., ubiquitin) encoding sequence between the splicing control module and the effector protein. Ubiquitin is cleaved through normal cellular processes, and so the Doublesex and Ubi amino acids are removed, leaving the effector protein (Bachmair et al., (1986) Science 234(4773): 179-186; Varshaysky, A. (2005) Meth. Enzymol. 399:777-799).

[0261] In some constructs, the effector protein tTAV or its homologs forms a positive feedback loop with the t/.sx-dcrivcd splice control module. Specifically, the (foe-derived splice control module was engineered to be under the control of a tetracycline responsive composite promoter, engineered by joining several (e.g., 7) repeats of TetO operator sequence from E. coli (TetO7) with a promoter (such as a minimal promoter from the heat shock protein 70 gene of Drosophila melanogaster (DmHsp70 minipro) (Gossen & Bujard, 1992; Gong et al., 2005)). The tTAV or its homologs then acts in a positive feedback loop as the binding of tTAV or its homologs to TetO drives further expression of that same protein. Without wishing to be bound by any particular theory of operation, it is believed that high level expression is deleterious to cells, likely due to transcriptional “squelching” (Gill and Ptashne, 1988). This feedback loop can be broken by the administration of tetracycline as this molecule, or analogue antibiotics are bound by tTAV which is thereby rendered unable to bind the operator, TetO.

[0262] The feedback loop operates specifically in females due to the addition splice control module (in any of the three Architectures discussed above) wherein the mRNAs produced in males and females are different due to sex-specific splicing. This, in turn means that the tTAV protein is only correctly encoded by an mRNA produced in females.

B. Preparation of the Vector Plasmid

[0263] Vector plasmids were prepared using the cloning vector pKC26-FB2 (Genbank #HQ998855). The plasmid backbone contains the pUC origin of replication and the betalactamase ampicillin resistance gene for use in molecular cloning procedures. This plasmid section is not included in the rDNA or incorporated into the insect genome. The vector plasmid also contains the complete rDNA constructs as shown in Table El A that is incorporated into the insect. The plasmids were prepared using routine DNA cloning procedures. C. Strain Generation

[0264] An. stephensi and An. Albimanus were reared under standard insectary conditions (26 °C, ±2 °C, 70% ±10% relative humidity and 12 h:12 h light:dark cycle). Mosquito embryos were transformed by standard micro-injection methods (Jasinskiene et al., (1998) Proc. Natl. ACad. Sci. USA 95:7520-7525; Morris, A. C. (1997) “Microinjection of mosquito embryos” In: Crampton, J. M„ Beard, C. B„ Louis, C. (Eds.), MOLECULAR BIOLOGY OE INSECT DISEASE VECTORS: A METHODS MANUAL. Chapman & Hall, 2-6 Boundary Row, London SEI 8HN, UK, pp. 423-429), injecting a combination of 75 ng/pl plasmid DNA and 250 ng/pl piggyBac mRNA, or 50 ng/pl plasmid DNA and 175 ng/pl piggyBac mRNA as the source of transposase. The plasmid DNA and the transposase mRNA were reconstituted in an injection buffer (5 mM KC1, 0.1 mM NaH2PO4, pH 6.8) made using standard laboratory grade reagents (Handler and James, 1998).

D. Strain Selection

[0265] Adult injection survivors (Generation 0 or Go) were back crossed to wildtype (WT). Gi pupae were screened for AmCyanl or DsRed2 fluorescence using a Leica M80 microscope equipped with filters for detection: maximum excitation 563 nm, emission 582 nm for DsRed2, and maximum excitation 458 nm, emission 489 nm for AmCyanl. G2 males from Gi families that express a fluorescent marker were crossed to WT females. Strains were maintained by crossing G3 males to Latin WT females. G4 hemizygous progeny from all strains were assessed for their survivability when reared in the presence and absence of antidote (doxycycline hyclate hydrochloride). The number of strains generated are shown in Table E1A.

[0266] Strains were assessed for the following traits: a) Male selection/female lethality: the effector protein coding gene, e.g. tTAV, needs to be effective in killing females in pre-adult life stages in the absence of the tetracycline analogue, at >90% effectiveness; b) Female repressibility: the lethal trait needs to be sufficiently repressible in the presence of a tetracycline analogue that females can be reared healthily and can lay fertile eggs, to enable production colonies to be maintained; c) Male tolerance of the lethal trait: the effector protein coding gene needs to be expressed in a sufficiently female sex-specific manner that it does not kill or impair male fitness significantly in the absence of the tetracycline analogue.

[0267] Not all strains tested satisfied all criteria. While not wishing to be bound by any particular theory, higher numbers of strains comprising a splice control module with Architecture 1 showed the assessed traits compared to those with Architecture 2 or 3. The location and size of genetic components in the transgene of each of the strains meeting the selection criteria are shown in Tables E1B-E1R and FIGs. 2A-2Q.

Table E1B. Location and size of genetic components in the 0X5624 transgene.

Table E1C. Location and size of genetic components in the 0X5650 transgene. Table EID. Location and size of genetic components in the 0X5654 transgene.

Table E1E. Location and size of genetic components in the 0X5676 transgene.

Table E1F. Location and size of genetic components in the 0X5678 transgene. Table E1G. Location and size of genetic components in the 0X5680 transgene.

Table E1H. Location and size of genetic components in the 0X5693 transgene.

Table Ell. Location and size of genetic components in the 0X5691 transgene.

Table El J. Location and size of genetic components in the 0X5718 transgene.

Table E1K. Location and size of genetic components in the 0X5720 transgene.

Table EIL. Location and size of genetic components in the 0X5677 transgene.

Table EIM. Location and size of genetic components in the 0X5679 transgene. Table EIN. Location and size of genetic components in the 0X5681 transgene.

Table E10. Location and size of genetic components in the 0X5719 transgene.

Table EIP. Location and size of genetic components in the 0X5721 transgene.

Table EIQ. Location and size of genetic components in the 0X5733 transgenes.

Table 1R. Location and size of genetic components in the 0X5734 transgenes.

Example 2 - Genetically Engineered Anopheles via Female-Specific Lethal Genes.

[0268] This examples demonstrates generation of male- selecting strains of Anopheles stephensi and An. albimanus using an alternative approach to alternative splicing, which is based on the lethal effect of female- specific lethal genes.

[0269] Genetically engineered Anopheles stephensi and An. albimanus strains were generated by insertion of one of the recombinant DNA (rDNA) constructs generated into the An. stephensi or An. Albimanus genome. The DNA constructs generally comprise two gene cassettes between the 5’ and 3’ fragments of the Trichoplusia ni piggyBac transposase used to insert them into the insect genome: (1) a first module comprising a coding sequence for tTAV or a variant thereof, and a second module comprising a sex-specific lethal gene (e.g., a femalespecific lethal gene) linked to a promoter repressible by tetracycline or by a tetracycline analogue, and (2) a color marker cassette for expressing DsRed2. The components comprised by the DNA constructs are listed in Table El A.

[0270] Two female- specific lethal genes YOB and GUY1 were designed and tested. For YOB, tTAV of the first module was under the control of bZipl, an early zygotic promoter, thus tTAV expression from very early embryonic development was achieved. In the absence of tetracyclines, tTAV can bind to the TRE3G promoter of the second module comprising 7 Tet operon repeats, and stimulate expression of the YOB protein (SEQ ID NOs: 28-29), which is expected to be lethal to female embryos. In this way, conditional female- specific lethality might be achievable without relying on alternative splicing. Two constructs were designed using different 3’ UTRs in the first module (FIG. 4A). The nucleic acid sequence of the YOB coding sequences is set forth in SEQ ID NO: 31. The amino acid sequence of the YOB protein is set forth is SEQ ID NO: 32.

[0271] Similarly for GUY1, a tTAV coding sequence is operably linked to an early zygotic promoter (GUY1 promoter, bZipl, or Vasa). In the absence of tetracyclines, tTAV will activate the expression of GUY1 by binding to TRE3G. As shown in FIG. 4B, different constructs were designed using different promoters of the first module and different 3’ UTRs. Additionally, one construct was made using TRE3G as the promoter for both modules. The nucleic acid sequence of the GUY 1 coding sequences is set forth in SEQ ID NO: 28. The amino acid sequence of the GUY1 protein is set forth is SEQ ID NO: 29. [0272] Together these gene cassettes deliver An. stephensi and An. albimanus strains that, when reared in the presence of a tetracycline analogue (e.g., tetracycline-class antibody), development occurs normally in both sexes, but when reared in the absence of tetracycline females do not survive to adulthood and a male-only cohort is produced. Additionally, each insect is marked with the fluorescent DsRed2 protein.

SEQUENCES

Claims

1. A doublesex (dsx) splice control module polynucleotide comprising, from 5’ to 3’: i. a dsx exon 4 sequence; ii. a dsx intron 4 sequence; iii. a first portion of a dsx exon 5 sequence; iv. an effector protein coding sequence; v. a second portion of the dsx exon 5 sequence; vi. a dsx intron 5 sequence; and vii. a dsx exon 6 sequence.

2. The dsx splice control module polynucleotide of claim 1, wherein the dsx splice control module polynucleotide is derived from an Anopheles dsx.

3. The dsx splice control module polynucleotide of claim 2, wherein the dsx splice control module polynucleotide is derived from an Anopheles stephensi (AsteDsx), Anopheles albimanus (AalhDsx). Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles coluzzii, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freeborni, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, or Anopheles gambiae dsx.

4. The dsx splice control module polynucleotide of claim 3, wherein the dsx splice control module polynucleotide is derived from Anopheles stephensi dsx (AsteDsx).

5. The dsx splice control module polynucleotide of claim 3, wherein the dsx splice control module polynucleotide is derived from Anopheles albimanus dsx (AalbDsx).

6. The dsx splice control module polynucleotide of any one of claims 1-5, wherein the first portion of the dsx exon 5 sequence comprises a 5’ terminal fragment of dsx exon 5.

7. The dsx splice control module polynucleotide of any one of claims 1-6, wherein the second portion of the dsx exon 5 sequence comprises a 3’ terminal fragment of dsx exon 5.

8. The dsx splice control module polynucleotide of any one of claims 1-7, wherein the effector protein is lethal, deleterious, or sterilizing to an insect.

9. The dsx splice control module polynucleotide of any one of claims 1-8, wherein expression of the effector protein causes lethality in pre-adult females in the absence of tetracycline or an analogue thereof, with at least 90% effectiveness.

10. The dsx splice control module polynucleotide of any one of claims 1-9, wherein the effector protein is selected from the group consisting of: a tTA or a tTAV gene variant, or a variant thereof, ReaperKR or a variant thereof, an apoptosis-inducing factor or a variant thereof, Hid or a variant thereof, and NipplDm or a variant thereof.

11. The dsx splice control module polynucleotide of claim 10, wherein the effector protein is tTAV, tTAV2, or tTAV3.

12. The dsx splice control module polynucleotide of claim 11, wherein the effector protein coding sequence comprises the sequence set forth in any of SEQ ID NOs: 18, 20, and 22.

13. The dsx splice control module polynucleotide of claim 10, wherein the effector protein coding sequence comprises the sequence set forth in any of SEQ ID NOs: 26, and 30.

14. The dsx splice control module polynucleotide of any one of claims 1-13, wherein the dsx exon 4 sequence is modified compared to a wild-type dsx exon 4 sequence and/or the first portion of the dsx exon 5 sequence is modified compared to a wild-type dsx exon 5 sequence to remove one or more start codons.

15. The dsx splice control module polynucleotide of any one of claims 1-14, wherein the dsx exon 4 sequence is modified compared to a wild-type dsx exon 4 sequence and/or the first portion of the dsx exon 5 sequence is modified compared to a wild-type dsx exon 5 sequence to remove one or more stop codons.

16. The dsx splice control module polynucleotide of claim 14 or 15, wherein the modified dsx exon 4 sequence and/or first portion of the dsx exon 5 comprises one or more modifications selected from the group consisting of a substitution, an insertion, and a deletion.

17. The dsx splice control module polynucleotide of any one of claims 1-16, wherein dsx splice control module polynucleotide does not comprise any open reading frame 5’ of the effector protein coding sequence.

18. The dsx splice control module polynucleotide of any one of claims 1-17, wherein the splice control module polynucleotide is spliced on a sex-specific basis when introduced into an insect to produce one or more female splice forms in female insects and one or more male splice forms in male insects.

19. The dsx splice control module polynucleotide of claim 18, wherein the female splice forms express the effector protein.

20. The dsx splice control module polynucleotide of claim 18, wherein the male splice form expresses the effector protein.

21. The dsx splice control module polynucleotide of claim 18, wherein the male splice form does not express the effector gene in significant quantities.

22. The dsx splice control module polynucleotide of claim 18, wherein the one or more female splice forms comprise, from 5’ to 3’: i. a dsx exon 4 sequence; ii. a first portion of a dsx exon 5 sequence; iii. an effector protein coding sequence; iv. a second portion of a dsx exon 5 sequence; and vi. a dsx exon 6 sequence.

23. The dsx splice control module polynucleotide of claim 19, wherein the splice control module is capable of producing a plurality of different female splice forms in female insects, and wherein the length of the first portion of the dsx exon 5 sequence is variable between the plurality of different female splice forms.

24. The dsx splice control module polynucleotide of any one of claims 1-23, wherein the splice control module further comprises a 3’ UTR sequence.

25. The dsx splice control module polynucleotide of claim 24, wherein the 3’ UTR sequence is derived from a PIO 3’ UTR or a SV40 3’ UTR.

26. The dsx splice control module polynucleotide of claim 25, wherein the 3’ UTR sequence is derived from PIO 3’ UTR comprising the sequence set forth in SEQ ID NO: 33.

27. The dsx splice control module polynucleotide of claim 25, wherein the 3’ UTR sequence is derived from SV40 3’ UTR comprising the sequence set forth in SEQ ID NO: 34.

28. A doublesex (dsx) splice control module polynucleotide operably linked to an effector protein coding sequence, comprising, from 5’ to 3’: i. a dsx exon 4 sequence; ii. a dsx intron 4 sequence; iii. a dsx exon 5 sequence; iv. a dsx intron 5 sequence; and v. a dsx exon 6 sequence, wherein the effector protein coding sequence is 3’ of the dsx splice control module polynucleotide.

29. The dsx splice control module polynucleotide of claim 28, wherein the dsx splice control module polynucleotide is derived from an Anopheles dsx.

30. The dsx splice control module polynucleotide of claim 28 or 29, wherein the effector protein is tTAV, tTAV2, or tTAV3.

31. The dsx splice control module polynucleotide of any one of claims 28-30, wherein the splice control module polynucleotide is spliced on a sex-specific basis when introduced into an insect to produce one or more female splice forms in female insects and one or more male splice forms in male insects.

32. The dsx splice control module polynucleotide of any one of claims 28-31, wherein the effector protein coding sequence comprises the sequence set forth in any of SEQ ID NOs: 18, 20, and 22.

33. A gene expression polynucleotide, comprising: a first module comprising from 5’ to 3’: i. a promoter operable in an insect; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR; and a second module comprising from 5’ to 3’: i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a female- specific lethal protein; and iv. a 3’ UTR.

34. The gene expression polynucleotide of claim 33, wherein the female-specific lethal protein is lethal to female insects.

35. The gene expression polynucleotide of claim 33 or 34, wherein the female- specific lethal protein is YOB or a variant thereof, or GUY 1 or a variant thereof.

36. The gene expression polynucleotide of any one of claims 33-35, wherein the femalespecific lethal protein comprises the sequence set forth in any of SEQ ID NOs: 29 and 32.

37. The gene expression polynucleotide of any one of claims 33-36, wherein the coding sequence of the female- specific lethal protein comprises the sequence set forth in any of SEQ ID NOs: 28 and 31.

38. The gene expression polynucleotide of any one of claims 33-37, wherein the promoter of the first module is selected from the group consisting of bZIPl, GUY1 promoter, Vasa, Tre3G, Drosophila melanogaster minimal HSP70 promoter (DmHsp70), CMV minipromoter, OpIE2, and Act5c.

39. The gene expression polynucleotide of any one of claims 33-38, wherein the first module further comprises a 5’ UTR, optionally wherein the 5’ UTR of the first module is derived from a GUY1 5’ UTR comprising the sequence set forth in SEQ ID NO: 66.

40. The gene expression polynucleotide of any one of claims 33-39, wherein the promoter of the first module comprises a sequence as found in any one of SEQ ID NOs: 36, 39, 40, and 65.

41. The gene expression polynucleotide of any one of claims 33-40, wherein the coding sequence of tTAV or a variant thereof comprises the sequence set forth in any of SEQ ID NOs: 18, 20, and 22.

42. The gene expression polynucleotide of any one of claims 33-41, wherein the 3’ UTR of the first module is derived from a K10 3’ UTR or a SV40 3’ UTR.

43. The gene expression polynucleotide of claim 42, wherein the 3’ UTR of the first module is derived from K10 3’ UTR comprising the sequence set forth in SEQ ID NO: 56.

44. The gene expression polynucleotide of claim 42, wherein the 3’ UTR of the first module is derived from SV40 3’ UTR comprising the sequence set forth in SEQ ID NO: 34.

45. The gene expression polynucleotide of any one of claims 33-44, wherein the promoter of the second module comprises a sequence as found in SEQ ID NO: 36.

46. The gene expression polynucleotide of any one of claims 33-45, wherein the 5’ UTR of the second module is derived from YOB 5’ UTR comprising the sequence set forth in SEQ ID NO: 67.

47. The gene expression polynucleotide of any one of claims 33-45, wherein the 5’ UTR of the second module is derived from GUY1 5’ UTR comprising the sequence set forth in SEQ ID NO: 66.

48. The gene expression polynucleotide of any one of claims 33-47, wherein the 3’ UTR of the second module is derived from PIO 3’ UTR comprising the sequence set forth in SEQ ID NO: 33.

49. The gene expression polynucleotide of any one of claims 33-47, wherein the 3’ UTR of the second module is derived from YOB 3’ UTR comprising the sequence set forth in SEQ ID NO: 68.

50. The gene expression polynucleotide of any one of claims 33-47, wherein the 3’ UTR of the second module is derived from GUY1 3’ UTR comprising the sequence set forth in SEQ ID NO: 69.

51. The gene expression polynucleotide of claim 33, comprising: a first module comprising from 5’ to 3’: i. a bZIPl promoter; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR selected from the group consisting of: SV40 and K10; and a second module comprising from 5’ to 3’: i. a TRE3G promoter; ii. a YOB 5’ UTR; iii. a YOB coding sequence; and iv. a 3’ UTR selected from the group consisting of: YOB and plO.

52. The gene expression polynucleotide of claim 33, comprising: a first module comprising from 5’ to 3’: i. a promoter selected from the group consisting of: bZIPl, GUY1, Vasa, and TRE3G; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR selected from the group consisting of: SV40 and K10; and a second module comprising from 5’ to 3’: i. a TRE3G promoter; ii. a GUY1 5’ UTR; iii. a GUY 1 coding sequence; and iv. a 3’ UTR selected from the group consisting of: GUY1 and plO.

53. The polynucleotide of any of claims 1-52, wherein the insect is of the Order Diptera.

54. The polynucleotide of any of claims 1-52, wherein the insect is a mosquito of a genus selected from the group consisting of Anopheles, Stegomyia, Aedes, and Culex.

55. The polynucleotide of claim 54, wherein the mosquito is a species selected from the group consisting of Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freeborni, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles arabiensis, Anopheles coluzzii, and Anopheles gambiae.

56. The polynucleotide of any of claims 1-52, wherein the insect is a Calliphoridae species selected from the group consisting of Cochliomyia hominivorax, Chrysomya bezz.iana. and Lucilia cuprina.

57. The polynucleotide of any of claims 1-52, wherein the insect is a Diptera of a species selected from the group consisting of Ceratitis capitata, Anastrepha ludens, Bactrocera dorsalis, Bactrocera oleae, Bactrocera cucurbitae, Ceratitis rosa, Rhagoletis cerasi, Bactrocera tyroni, Bactrocera zonata, Anastrepha suspense, and Anastrepha obliqua.

58. A gene expression system comprising the dsx splice control module polynucleotide of any of claims 1-32.

59. The gene expression system of claim 58, further comprising a 5’ untranslated region (5’ UTR) operably linked 5’ of the dsx splice control module polynucleotide.

60. The gene expression system of claim 59, further comprising a promoter operable in an insect.

61. The gene expression system of claim 59, wherein the promoter is selected from the group consisting of Drosophila melanogaster minimal HSP70 promoter (DmHsp70), Tre3G, CMV minipromoter, OpIE2, Vasa, bZIPl, and Act5c.

62. The gene expression system of any of claims 59-61, wherein the gene expression system comprises a UTR sequence as found in any one of SEQ ID NOs: 35-41.

63. The gene expression system of any of claims 60-62, wherein the promoter comprises a sequence set forth in any one of SEQ ID NOs: 42-44.

64. The gene expression system of any of claims 58-63, further comprising a tetracycline responsive operator.

65. An expression vector plasmid comprising the gene expression system of any of claims 58-64 or the gene expression polynucleotide of any of claims 33-52.

66. The expression vector plasmid of claim 65, further comprising a polynucleotide encoding a color marker protein.

67. The expression vector plasmid of claim 66, wherein the color marker protein is a fluorescent marker protein.

68. The expression vector plasmid of claim 67, wherein the fluorescent marker protein is GFP, ZsGreenl, mCherry, DsRed-Monomer, DsRed-Express, DsRed-Express2, tdTomato, AsRed2, mStrawberry, E2-Crimson, HcRedl, mRaspberry, mPlum, m0range2, mBanana, ZsYellowl, Dendra2, PAmCherry, Timer, DsRed2, or AmCyanl.

69. The expression vector plasmid of any of claims 66-68, wherein the polynucleotide encoding the fluorescent marker protein is operably linked to a promoter.

70. The expression vector plasmid of claim 69, wherein the promoter is an IE1 promoter or a 3xP3 promoter.

71. The expression vector plasmid of any of claims 66-70, further comprising an enhancer.

72. The expression vector plasmid of claim 71, wherein the enhancer is Hr5 enhancer.

73. The expression vector plasmid of claim 71 or 72, wherein the enhancer is truncated.

74. The expression vector plasmid of any of claims 65-73, wherein the expression vector plasmid is introduced into the genome of an insect using piggyBac transposable elements, Mariner transposable elements, PhiC31, ZFNs, TALENs, or CRISPR.

75. The expression vector plasmid of any of claims 65-74, comprising a sequence that is at least 80%, 85%, 90%, or 95% identical to any one of SEQ ID NOs: 1-17.

76. The expression vector plasmid of claim 75, comprising a sequence set forth in any one of SEQ ID NOs: 1-17.

77. A genetically engineered insect comprising a gene expression system incorporated into a chromosome of the insect, the gene expression system comprising: i. a doublesex (dsx') splice control module wherein the dsx splice control module comprises the components from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a first portion of a dsx exon 5 sequence; iv) an effector protein coding sequence; v) a second portion of a dsx exon 5 sequence; vi) a dsx intron 5; and vii) a dsx exon 6; and ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect.

78. A genetically engineered insect comprising a gene expression system incorporated into a chromosome of the insect, the gene expression system comprising: i. a doublesex (dsx) splice control module polynucleotide operably linked to an effector protein coding sequence, wherein the dsx splice control module comprises, from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a dsx exon 5 sequence; iv) a dsx intron 5 sequence; and v) a dsx exon 6 sequence; wherein the effector protein coding sequence is 3’ of the dsx splice control module polynucleotide; ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the effector protein coding sequence; and iv. a promoter operable in the insect.

79. The genetically engineered insect of claim 77 or 78, wherein the dsx splice control module is derived from an Anopheles dsx.

80. A genetically engineered insect comprising a gene expression system incorporated into a chromosome of the insect, the gene expression system comprising: a first module comprising from 5’ to 3’: i. a promoter operable in the insect; ii. a coding sequence of tTAV or a variant thereof; ii. a 3’ UTR; and a second module comprising from 5’ to 3’: i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a female- specific lethal protein; and iv. a 3’ UTR.

81. The genetically engineered insect of any one of claims 77-80, wherein the insect is a mosquito.

82. The genetically engineered insect of claim 81, wherein the mosquito is a mosquito of the genus Anopheles, Aedes, Stegomyia or Culex.

83. The genetically engineered insect of claim 82, wherein the mosquito is Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles culicifacies, Anopheles dims, Anopheles far auti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles arabiensis, Anopheles coluzzii, or Anopheles gambiae.

84. A female genetically engineered insect comprising a gene expression system incorporated into a chromosome of the insect, wherein the gene expression system comprises: i. a doublesex (dsx') splice control module wherein the splice control module is capable to produce a splice form comprising the components from 5’ to 3’: i) a dsx exon 4 sequence; iii) a first portion of a dsx exon 5 sequence; iv) an effector protein coding sequence; v) a second portion of a dsx exon 5 sequence; and vii) a dsx exon 6; wherein the dsx splice control module is spliced specifically in the female genetically engineered insect to produce one or more female splice forms that express the effector protein; and ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect.

85. The female genetically engineered insect of claim 84, wherein the effector protein is lethal, deleterious, or sterilizing to the insect.

86. The genetically engineered insect of any of claims 77-80, wherein the gene expression system further comprises a polynucleotide encoding a fluorescent protein.

87. The genetically engineered insect of claim 86, wherein the polynucleotide encoding the fluorescent protein is operably linked to a promoter.

88. The genetically engineered insect of claim 87, wherein the fluorescent protein is selected from the group consisting of GFP, ZsGreenl, mCherry, DsRed- Monomer, DsRed- Express, DsRed-Express2, tdTomato, AsRed2, mStrawberry, E2-Crimson, HcRedl, mRaspberry, mPlum, mOrange2, mBanana, ZsYellowl, Dendra2, PAmCherry, Timer, DsRed2, and AmCyanl, and the promoter is an IE1 promoter or a 3xP3 promoter.

89. A method of producing a genetically engineered insect comprising inserting a gene expression system into the insect’s genome, wherein the gene expression system comprises: i. a doublesex (dsx) splice control module wherein the splice control module comprises the components from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a first portion of a dsx exon 5 sequence; iv) an effector protein coding sequence; v) a second portion of a dsx exon 5 sequence; vi) a dsx intron 5; and vii) a dsx exon 6; ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the splice control module; and iv. a promoter operable in the insect; or wherein the gene expression system comprises: i. a doublesex (dsx) splice control module polynucleotide operably linked to an effector protein coding sequence, wherein the dsx splice control module comprises, from 5’ to 3’: i) a dsx exon 4 sequence; ii) a dsx intron 4 sequence; iii) a dsx exon 5 sequence; iv) a dsx intron 5 sequence; and v). a dsx exon 6 sequence; wherein the effector protein coding sequence is 3’ of the dsx splice control module polynucleotide; ii. a 5’ UTR positioned 5’ of the splice control module; iii. a 3’ UTR positioned 3’ of the effector protein coding sequence; and iv. a promoter operable in the insect.

90. The method of claim 89, wherein the dsx splice control module is derived from an Anopheles dsx.

91. A method of producing a genetically engineered insect comprising inserting a gene expression system into the insect’s genome, wherein the gene expression system comprises: a first module comprising from 5’ to 3’: i. a promoter operable in the insect; ii. a coding sequence of tTAV or a variant thereof; iii. a 3’ UTR; and a second module comprising from 5’ to 3’: i. a promoter repressible by tetracycline or by a tetracycline analogue; ii. a 5’ UTR; iii. a coding sequence of a female- specific lethal protein; and iv. a 3’ UTR.

92. The method of any of claims 89-91, wherein the insect is a mosquito.

93. The method of claim 92, wherein the mosquito is a mosquito of the genus Anopheles, Aedes, Stegomyia or Culex.

94. The method of claim 93, wherein the mosquito is Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles culicifacies, Anopheles dirus, Anopheles farauti, Anopheles flavirostris, Anopheles fluviatilis, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles arabiensis, Anopheles coluzzii, or Anopheles gambiae.

95. The method of claim 94, wherein the gene expression system further comprises a polynucleotide encoding a fluorescent protein.

96. The method of claim 95, wherein the polynucleotide encoding the fluorescent protein is operably linked to a promoter.

97. The method of claim 96, wherein the fluorescent protein is selected from the group consisting of GFP, ZsGreenl, mCherry, DsRed-Monomer, DsRed-Express, DsRed-Express2, tdTomato, AsRed2, mStrawberry, E2-Crimson, HcRedl, mRaspberry, mPlum, m0range2, mBanana, ZsYellowl, Dendra2, PAmCherry, Timer, DsRed2, and AmCyanl, and the promoter is an IE1 promoter or a 3xP3 promoter.

98. A method of selectively rearing male genetically engineered insects comprising, rearing a genetically engineered insect of any of claims 77-88, wherein the rearing is in the absence of tetracycline or an analogue thereof.

99. A male genetically engineered male insect produced by the method of claim 98.

100. A method of reducing a wild insect population comprising contacting the wild insect population with a plurality of the male genetically engineered insects of claim 99, wherein the male genetically engineered insects mate with wild female insects.

101. The method of claim 100, wherein the insect is a mosquito.

102. The method of claim 101, wherein the mosquito is a mosquito of the genus Anopheles, Aedes, Stegomyia or Culex.

103. The method of claim 102, wherein the mosquito is Anopheles stephensi, Anopheles albimanus, Anopheles annularis, Anopheles anthropophagus, Anopheles arabiensis, Anopheles aquasalis, Anopheles atroparvus, Anopheles barbirostris, Anopheles culicifacies, Anopheles dims, Anopheles farauti, Anopheles flavirostris, Anopheles fluvial ills, Anopheles freebomi, Anopheles funestus, Anopheles labranchiae, Anopheles maculatus, Anopheles darlingi, Anopheles marajoara, Anopheles melas, Anopheles messeae, Anopheles minimus, Anopheles multicolor, Anopheles nunez-tovari, Anopheles punctulatus group, Anopheles pharoahensis, Anopheles pseudopunctipennis, Anopheles pulcherrimus, Anopheles quandrimaculatus, Anopheles sacharovi, Anopheles sergentii, Anopheles sinensis, Anopheles sundaicus, Anopheles superpictus, Anopheles arabiensis, Anopheles coluzzii, or Anopheles gambiae.