Systems, compositions, and methods for generating cisgenic sexing strains in insects

Abstract

Provided herein are methods of generating a sex-distinguishable insect population that include (a) delivering a cisgenic nucleic acid molecule into an insect genome, wherein the nucleic acid molecule comprises a sex-specific splicing module, and wherein the nucleic acid molecule is introduced into an endogenous phenotypic gene of the insect; (b) generating the sex-distinguishable insect population, wherein an insect of the sex-distinguishable insect population comprises the cisgenic nucleic acid molecule; and (c) sorting the insect from the sex-distinguishable insect population based on the differential sex-specific expression of the endogenous phenotypic gene, wherein the endogenous phenotypic gene is functionally expressed in only one sex.

Description

[0001] Atorney Docket No.: 15670-0446WO1

[0002] SYSTEMS, COMPOSITIONS, AND METHODS FOR GENERATING CISGENIC SEXING STRAINS IN INSECTS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority’ to U.S. Provisional Patent Application No.

[0004] 63 / 738,236, filed on December 23, 2024. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated herein by reference in its entirety.

[0005] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under AP24PPQST00C152 awarded by the U.S. Department of Agriculture. The government has certain rights in the invention.

[0007] SEQUENCE LISTING

[0008] This application contains a Sequence Listing that has been submitted electronically as an XML file named “15670-0446W01_ST26_SL.xml.” The XML file, created on December 18, 2025, is 11,852 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0009] BACKGROUND

[0010] Genetic sexing strains (GSSs) have been developed in multiple insect species of economic significance to allow easier male and female separation necessary for efficient population control. Specifically, GSSs are used within sterile insect technique (SIT) programmes which work via frequent releases of sterilised insects into the wild for temporary population control. Male-only releases, aided by GSS implementation, strongly succour in released fly dispersal and their mating frequency with wild females, thus enhancing SIT success. Vast efforts have focused on GSS generation in mosquito disease vectors and agricultural fruit fly pests to avoid labour-intensive sex-sorting by eliminating the females from the released population early in the life cycle.Atorney Docket No.: 15670-0446WO1

[0011] SUMMARY

[0012] Provided herein are methods of generating a sex-distinguishable insect population that include (a) delivering a cisgenic nucleic acid molecule into an insect genome, wherein the nucleic acid molecule comprises a sex-specific splicing module, and wherein the nucleic acid molecule is introduced into an endogenous phenotypic gene of the insect; (b) generating the sex-distinguishable insect population, wherein an insect of the sex-distinguishable insect population comprises the cisgenic nucleic acid molecule; and (c) sorting the insect from the sex-distinguishable insect population based on the differential sex-specific expression of the endogenous phenotypic gene, wherein the endogenous phenotypic gene is functionally expressed in only one sex.

[0013] In some embodiments, the sex-specific splicing module activates the expression of the endogenous phenotypic gene. In some embodiments, the sex-specific splicing module disrupts the expression of the endogenous phenotypic gene. In some embodiments, the sexspecific splicing module comprises an endogenous sex-specific intronic sequence. In some embodiments, the endogenous sex-specific intronic sequence comprises an endogenous transformer (tra) intron. In some embodiments, the endogenous phenotypic gene comprises a white pupae (wp) gene.

[0014] In some embodiments, the cisgenic nucleic acid molecule is introduced into the endogenous phenotypic gene of the insect by using a gene-editing agent, transposase-mediated insertion, HDR replacement, or recombinase-mediated cassette exchange. In some embodiments, the gene-editing agent is selected from the group consisting of a CRISPR / Cas9 system, a CRISPR / Casl2 system, a Cast 3 system, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), homology-directed repair, non-homologous end-joining (NHEJ) repair, and recombinases. In some embodiments, the geneediting agent comprises CRISPR / Cas9 components. In some embodiments, the sex-distinguishable insect population is generated by cisgenic genetic modification. In some embodiments, the cisgenic nucleic acid molecule is delivered into the insect at the embryo stage of development of the insect.

[0015] In some embodiments, the insect is sorted as male based on the expression of the endogenous phenotypic gene. In some embodiments, the insect is sorted as female based on the expression of the endogenous phenotypic gene. In some embodiments, the differential sex-specific expression of the endogenous phenotypic gene comprises molecular detection, behavioral phenoty pe, viability differences, or size or timing differences. In someAtorney Docket No.: 15670-0446WO1

[0016] embodiments, the insect is sorted by automated machine-vision sorting, fluorescence-based sorting, or mechanical sex-separation enhanced by phenotype. In some embodiments, the insect is sorted at the larval stage, the pupal developmental stage, or the adult stage.

[0017] In some embodiments, the insect is a mosquito or a fruit fly. In some embodiments, the insect is a tephritid fruit fly selected from Medfly (Ceratitis capitata), Mexfly (Anastrepha ludens), Oriental fruit fly (Bactrocera dorsalis), Olive fruit fly (Bactrocera oleae), Melon fly (Bactrocera cucurbitae). Natal fruit fly (Ceratitis rosa), Cherry fruit fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyrom). Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), Oriental Fruit Fly (Bactrocera dorsalis), West Indian fruit fly (Anastrepha obliqua), the New World screwworm (Cochliomyia hominivorax), the Old World screwworm (Chrysomya bezziana), Australian sheep blowfly / greenbottle fly (Lucilia cuprina), the pink bollworm (Pectinophora gossypiella), the European Gypsy moth (Lymantria dispar), the Navel Orange Worm (Amyelois transitella), the Peach Twig Borer (Anarsia lineatella), the rice stem borer (Tryporyza incertulas), the noctuid moths, Heliothinae, the Japanese beetle (Papilla japonica), White-fringed beetle (Graphognatus spp.), Boll weevil (Anthonomous grandis), the Colorado potato beetle (Leptinotarsa decern lineata), the vine mealybug (Pianococcus ficus), Asian citrus psyllid (diaphorina citri), Spotted wing drosophila (drosophila suzukii), Bluegreen sharpshooter (graphocephala atropunctata), Glassy winged sharpshooter (Flomalodisca vitripennis), Light brown apple moth (Epiphyas postvittana), Bagrada bug (Bagrada hilaris), Brown marmorated stink bug (Halyomorpha halys), Asian Gypsy Moth selected from the group of Lymantria dispar asiatica, Lymantria dispar japonica, Lymantria albescens, Lymantria umbrosa, and Lymantria postalba, Asian longhomed beetle (Anoplophora glabripennis), Coconut Rhinoceros Beetle (Oryctes rhinoceros), Emerald Ash Borer (Agrilus planipennis), European Grapevine Moth (lobesia botrana), European Gypsy Moth (Lymantria dispar). False Codling Moth (Thaumatotibialeucotreta), fire ants selected from Solenopsis invicta Buren, and S. richteri Forel, Old World Bollworm (Flelicoverpa armigera), Spotted Lantemfly (Lycorma delicatula), Africanized honeybee (apis mellifera scutellata), Fruit and shoot borer (leucinodes orbonalis), com root worm (Diabrotica spp.). Western com rootworm (diabrotica virgifera). Whitefly (bemisia tabaci), Flouse Fly (Musca Domestica), Green Bottle Fly (Lucilia cuprina), Silk Moth (Bombyx mori), Red Scale (Aonidiella aurantia), Dog heartworm (Dirofilaria immitis), Southern pine beetle (Dendroctonus frontalis), Avocado thrip (Thysanoptera Spp.), Botfly selected from Oestridae spp. and Dermatobia hominis), Florse Fly (Tabanus sulcifrons), Flom Fly (Flaematobia irritans), Screwworm Fly selectedAtorney Docket No.: 15670-0446WO1

[0018] from Cochliomyia macellaria (C. macellaria), C. hominivorax, C. aldrichi, or C. minima. Tsetse Fly (Glossina spp.). Warble Fly selected from Flypoderma bovis or Hypoderma lineatum, Spoted lantemfly (Lycorma delicatula), Khapra beetle (Trogoderma granarium), Honeybee mite (Varroa destructor), Termites (Coptotermes formosanus), Hemlock woolly adelgid (Adelges tsugae), Walnut twig beetle (Pityophthorus juglandis), European wood wasp (Sirex noctilio), Pink-spoted bollworm (pectinophora scutigera), Two spoted spider mite (Tertanychus urticae), Diamondback moth (plutella xylostella). Taro caterpillar (spodoptera litura), Red flour beetle (tribolium castaneum), Green peach aphid (Myzus persicae), Coton Aphid (aphis gossypii), Brown planthopper (nilaparvata lugens), Beet armyworm (spodotera exigua), Western flower thrips (frankliniella occidentalis), Codling moth (cydia pomonella), Cowpea weevil (callosobruchus maculatus). Pea aphid (acyrthosiphon pisum), Tomato leafminer (tuta absoluta). Onion thrips (thrips tabaci), or Cotton bollworm (Helicoverpa armigera). In some embodiments, the insect is a Ceratitis capitata (Mediterranean fruit fly). In some embodiments, the insect is a mosquito selected from the group consisting of Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus (Aedes triseriatus), Anopheles stephensi, Anopheles albimanus. Anopheles gambiae, Anopheles quadrimaculatus, Anopheles freebomi, Culex species, and Culisetamelanura.

[0019] Also provided herein are methods of identifying the sex of an insect based on a differential sex-specific phenotype that include (a) delivering a cisgenic nucleic acid molecule into an insect genome, wherein the nucleic acid molecule comprises a sex-specific splicing module, and wherein the nucleic acid molecule is introduced into an endogenous phenotypic gene of the insect; (b) generating a sex-distinguishable insect population, wherein an insect of the sex-distinguishable insect population comprises the cisgenic nucleic acid molecule; and (c) sorting the insect from the sex-distinguishable insect population based on the differential sex-specific expression of the endogenous phenotypic gene, wherein the endogenous phenotypic gene is functionally expressed in only one sex.

[0020] In some embodiments, the sex-specific splicing module activates the expression of the endogenous phenotypic gene. In some embodiments, the sex-specific splicing module disrupts the expression of the endogenous phenotypic gene. In some embodiments, the sexspecific splicing module comprises an endogenous sex-specific intronic sequence. In some embodiments, the endogenous sex-specific intronic sequence comprises an endogenous transformer (tra) intron. In some embodiments, the endogenous phenotypic gene comprises a white pupae (wp) gene.Atorney Docket No.: 15670-0446WO1

[0021] In some embodiments, the cisgenic nucleic acid molecule is introduced into the endogenous phenotypic gene of the insect by using a gene-editing agent, transposase-mediated insertion, HDR replacement, or recombinase-mediated cassette exchange. In some embodiments, the gene-editing agent is selected from the group consisting of a CRISPR / Cas9 system, a CRISPR / Casl2 system, a Casl3 system, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs). homology-directed repair, non-homologous end-joining (NHEJ) repair, and recombinases. In some embodiments, the geneediting agent comprises CRISPR / Cas9 components. In some embodiments, the sex-distinguishable insect population is generated by cisgenic genetic modification. In some embodiments, the cisgenic nucleic acid molecule is delivered into the insect at the embry o stage of development of the insect.

[0022] In some embodiments, the insect is sorted as male based on the expression of the endogenous phenotypic gene. In some embodiments, the insect is sorted as female based on the expression of the endogenous phenotypic gene. In some embodiments, the differential sex-specific expression of the endogenous phenotypic gene comprises molecular detection, behavioral phenotype, viability differences, or size or timing differences. In some embodiments, the insect is sorted by automated machine-vision sorting, fluorescence-based sorting, or mechanical sex-separation enhanced by phenotype. In some embodiments, the insect is sorted at the larval stage, the pupal developmental stage, or the adult stage.

[0023] In some embodiments, the insect is a mosquito or a fruit fly. In some embodiments, the insect is a tephritid fruit fly selected from Medfly (Ceratitis capitata), Mexfly (Anastrepha ludens), Oriental fruit fly (Bactrocera dorsalis), Olive fruit fly (Bactrocera oleae), Melon fly (Bactrocera cucurbitae), Natal fruit fly (Ceratitis rosa), Cherry' fruit fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyroni), Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa). Oriental Fruit Fly (Bactrocera dorsalis). West Indian fruit fly (Anastrepha obliqua), the New World screwworm (Cochliomyia hominivorax), the Old World screwworm (Chrysomya bezziana), Australian sheep blowfly / greenbottle fly (Lucilia cuprina), the pink bollworm (Pectinophora gossypiella), the European Gypsy moth (Lymantria dispar), the Navel Orange Worm (Amyelois transitella), the Peach Twig Borer (Anarsia lineatelia), the rice stem borer (Tryporyza incertulas), the noctuid moths, Heliothinae, the Japanese beetle (Papilla japonica), White-fringed beetle (Graphognatus spp.). Boll weevil (Anthonomous grandis), the Colorado potato beetle (Leptinotarsa decern lineata), the vine mealybug (Pianococcus ficus). Asian citrus psyllid (diaphorina citri).

[0024] Spotted wing drosophila (drosophila suzukii), Bluegreen sharpshooter (graphocephalaAtorney Docket No.: 15670-0446WO1

[0025] atropunctata), Glassy winged sharpshooter (Flomalodisca vitripennis), Light brown apple moth (Epiphyas postvittana), Bagrada bug (Bagrada hilaris), Brown marmorated stink bug (Halyomorpha halys), Asian Gypsy Moth selected from the group of Lymantria dispar asiatica, Lymantria dispar japonica, Lymantria albescens, Lymantria umbrosa, and Lymantria postalba, Asian longhomed beetle (Anoplophora glabripennis), Coconut Rhinoceros Beetle (Oryctes rhinoceros), Emerald Ash Borer (Agrilus planipennis), European Grapevine Moth (lobesia botrana), European Gypsy Moth (Lymantria dispar), False Codling Moth (Thaumatotibialeucotreta), fire ants selected from Solenopsis invicta Buren, and S. richteri Forel, Old World Bollworm (Flelicoverpa armigera), Spotted Lantemfly (Lycorma delicatula), Africanized honeybee (apis mellifera scutellata), Fruit and shoot borer (leucinodes orbonalis). com root worm (Diabrotica spp.), Western com rootworm (diabrotica virgifera). Whitefly (bemisia tabaci), Flouse Fly (Musca Domestica), Green Bottle Fly (Lucilia cuprina), Silk Moth (Bombyx mori), Red Scale (Aonidiella aurantia), Dog heartworm (Dirofilaria immitis), Southern pine beetle (Dendroctonus frontalis), Avocado thrip (Thysanoptera Spp.), Botfly selected from Oestridae spp. and Dermatobia hominis), Florse Fly (Tabanus sulcifrons), Flom Fly (Flaematobia irritans). Screwworm Fly selected from Cochliomyia macellaria (C. macellaria), C. hominivorax, C. aldrichi, or C. minima, Tsetse Fly (Glossina spp.), Warble Fly selected from Flypoderma bovis or Hypoderma lineatum. Spotted lantemfly (Lycorma delicatula), Khapra beetle (Trogoderma granarium), Honeybee mite (Varroa destructor), Termites (Coptotermes formosanus). Hemlock woolly adelgid (Adelges tsugae). Walnut twig beetle (Pityophthorus juglandis), European wood wasp (Sirex noctilio), Pink-spotted bollworm (pectinophora scutigera), Two spotted spider mite (Tertanychus urticae), Diamondback moth (plutella xylostella), Taro caterpillar (spodoptera litura), Red flour beetle (tribolium castaneum), Green peach aphid (Myzus persicae). Cotton Aphid (aphis gossypii), Brown planthopper (nilaparvata lugens), Beet armyworm (spodotera exigua), Western flower thrips (frankliniella occidentalism Codling moth (cydia pomonella), Cowpea weevil (callosobruchus maculatus), Pea aphid (acyrthosiphon pisum), Tomato leafminer (tuta absoluta), Onion thrips (thrips tabaci), or Cotton bollworm (Helicoverpa armigera). In some embodiments, the insect is a Ceratitis capitata (Mediterranean fruit fly). In some embodiments, the insect is a mosquito selected from the group consisting of Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus (Aedes triseriatus), Anopheles stephensi, Anopheles albimanus, Anopheles gambiae, Anopheles quadrimaculatus, Anopheles freebomi, Culex species, and Culiseta melanura.Atorney Docket No.: 15670-0446WO1

[0026] Also provided herein are cisgenic genetically modified insects comprising a nucleic acid molecule comprising a sex-specific splicing module within an endogenous phenotypic gene of the insect. In some embodiments, the sex-specific splicing module activates the expression of the phenotypic gene. In some embodiments, the sex-specific splicing module disrupts the expression of the phenotypic gene. In some embodiments, the sex-specific splicing module comprises an endogenous sex-specific intronic sequence. In some embodiments, the endogenous sex-specific intronic sequence comprises an endogenous transformer (tra) intron. In some embodiments, the phenotypic gene comprises a white pupae (wp) gene.

[0027] In some embodiments, the insect is sorted as male based on the expression of the phenotypic gene. In some embodiments, the insect is sorted as female based on the expression of the phenotypic gene.

[0028] In some embodiments, the insect is a mosquito or a fruit fly. In some embodiments, the insect is a tephritid fruit fly selected from Medfly (Ceratitis capitata), Mexfly (Anastrepha ludens), Oriental fruit fly (Bactrocera dorsalis), Olive fruit fly (Bactrocera oleae), Melon fly (Bactrocera cucurbitae). Natal fruit fly (Ceratitis rosa), Cherry fruit fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyrom). Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), Oriental Fruit Fly (Bactrocera dorsalis), West Indian fruit fly (Anastrepha obliqua), the New World screwworm (Cochliomyia hominivorax), the Old World screwworm (Chrysomya bezziana), Australian sheep blowfly / greenbottle fly (Lucilia cuprina), the pink bollworm (Pectinophora gossypiella), the European Gypsy moth (Lymantria dispar), the Navel Orange Worm (Amyelois transitella), the Peach Twig Borer (Anarsia lineatella), the rice stem borer (Tryporyza incertulas), the noctuid moths, Heliothinae, the Japanese beetle (Papilla japonica), White-fringed beetle (Graphognatus spp.), Boll weevil (Anthonomous grandis), the Colorado potato beetle (Leptinotarsa decern lineata), the vine mealybug (Pianococcus ficus), Asian citrus psyllid (diaphorina citri), Spotted wing drosophila (drosophila suzukii), Bluegreen sharpshooter (graphocephala atropunctata), Glassy winged sharpshooter (Flomalodisca vitripennis), Light brown apple moth (Epiphyas postvittana), Bagrada bug (Bagrada hilaris), Brown marmorated stink bug (Halyomorpha halys), Asian Gypsy Moth selected from the group of Lymantria dispar asiatica, Lymantria dispar japonica, Lymantria albescens, Lymantria umbrosa, and Lymantria postalba, Asian longhomed beetle (Anoplophora glabripennis), Coconut Rhinoceros Beetle (Oryctes rhinoceros), Emerald Ash Borer (Agrilus planipennis), European Grapevine Moth (lobesia botrana), European Gypsy Moth (Lymantria dispar), False Codling MothAtorney Docket No.: 15670-0446WO1

[0029] (Thaumatotibia leucotreta), fire ants selected from Solenopsis invicta Buren, and S. richteri Forel, Old World Bollworm (Flelicoverpa armigera), Spotted Lantemfly (Lycorma delicatula). Africanized honeybee (apis mellifera scutellata), Fruit and shoot borer (leucinodes orbonalis), com root worm (Diabrotica spp.), Western com rootworm (diabrotica virgifera), Whitefly (bemisia tabaci), Flouse Fly (Musca Domestica), Green Bottle Fly (Lucilia cuprina), Silk Moth (Bombyx mori), Red Scale (Aonidiella aurantia), Dog heartworm (Dirofilaria immitis). Southern pine beetle (Dendroctonus frontalis), Avocado thrip (Thysanoptera Spp.), Botfly selected from Oestridae spp. and Dermatobia hominis), Florse Fly (Tabanus sulcifrons), Flom Fly (Flaematobia irritans), Screwworm Fly selected from Cochliomyia macellaria (C. macellaria), C. hominivorax, C. aldrichi, or C. minima, Tsetse Fly (Glossina spp.). Warble Fly selected from Flypoderma bovis or Hypoderma lineatum, Spotted lantemfly (Lycorma delicatula), Khapra beetle (Trogoderma granarium), Honeybee mite (Varroa destructor), Termites (Coptotermes formosanus), Hemlock woolly adelgid (Adelges tsugae), Walnut twig beetle (Pityophthorus juglandis), European wood wasp (Sirex noctilio), Pink-spotted bollworm (pectinophora scutigera), Two spotted spider mite (Tertanychus urticae), Diamondback moth (plutella xylostella). Taro caterpillar (spodoptera litura), Red flour beetle (tribolium castaneum), Green peach aphid (Myzus persicae), Cotton Aphid (aphis gossypii), Brown planthopper (nilaparvata lugens), Beet armyworm (spodotera exigua), Western flower thrips (frankliniella occidentalis), Codling moth (cydia pomonella), Cowpea weevil (callosobruchus maculatus). Pea aphid (acyrthosiphon pisum), Tomato leafminer (tuta absoluta). Onion thrips (thrips tabaci), or Cotton bollworm (Helicoverpa armigera). In some embodiments, the insect is a Ceratitis capitata (Mediterranean fruit fly). In some embodiments, the insect is a mosquito selected from the group consisting of Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus (Aedes triseriatus), Anopheles stephensi, Anopheles albimanus. Anopheles gambiae, Anopheles quadrimaculatus, Anopheles freebomi, Culex species, and Culisetamelanura.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.Atorney Docket No.: 15670-0446WO1

[0031] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0032] BRIEF DESCRIPTION OF DRAWINGS FIGs. 1A-1B show an exemplary schematic of the design behind the cisgenic genetic sexing strain (CGSS). FIG. 1A shows a simplified diagram showcasing the CGSS strain generation and its underlying mechanism. The knock-in, mediated via homology-directed repair was performed into the Benakeion wild-type strain. Due to the presence of premature stop codons in the male-specific exons, the males are phenotypically white-pupaed, whilst in females, gene rescue occurs resulting in a brown-pupae phenotype (E1-E4, Exons 1-4; LHA, left homology7arm; MFS, Major Facilitator Superfamily; RHA, right homology' arm; tra, transformer, ' wp, white pupae). FIG. IB shows a graphic summary of the CGSS strain establishment via outcrosses to the irradiation-generated homozygous recessive white pupae mutant (wp- / -) strain (KI, knock-in).

[0033] FIGs. 2A-2D show characterization of the cisgenic genetic sexing strain (CGSS). FIG. 2A shows stack graphs displaying pupal colour and adult phenoty pes in the CGSS and VIENNA 8 strains for five consecutive generations (F2-F6). Chi-squared test significance levels are indicated as follows: p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***. FIG. 2B shows bar charts showing egg-adult survival of the CGSS strain compared to both it parental wild-type Benakeion, and VIENNA 8 strains, completed in biological triplicates. Egg-adult survival was measured using 5-hour collections of eggs and their subsequent hatching rates, hatched larval-pupal recovery rates, and pupal-adult recovery rates. Bar levels represent mean values, whilst individual replicate values are shown with dots. Dunn’s test significance levels are indicated as follows: p < 0.05 = *, p < 0.01 = **. FIG. 2C shows survival curves (Kaplan-Meier) of adult males and females from the CGSS strain, VIENNA 8 and wild-type Benakeion strains. The 95% confidence intervals are displayed using pale shading for each test group. FIG. 2D shows proportion of eclosing males and females from CGSS, VIENNA 8 and wild-ty pe Benakeion strains measured daily from age-matched triplicate 24-hour egg collections. Dots represent mean values of the replicates and standard error is indicated using whiskers.

[0034] FIG. 3 shows Oxford Nanopore genome sequencing of the white pupae genomic site, where diagrams depict the nanopore sequencing reads (top grey) aligning to the white pupae genomic location with the tra intron inserted in the CGSS strain.Atorney Docket No.: 15670-0446WO1

[0035] FIGs. 4A-4B show S2 white pupae is spliced sex-specifically in the CGSS strain. FIG. 4A shows a diagram showing the white pupae gene in the genome of the transformer (tra) intron CGSS knock-in (KJ) strain with forward and reverse primers labelled as F and R, accordingly (Table 1). FIG. 4B shows the annotated electrophoresis gel of the PCR products using primers from FIG.4A. The PCR was performed on genomic and complementary DNA templates from wild-type and CGSS adults. The DNA ladder and the negative control were run in the first and tenth wells, respectively.

[0036] FIG. 5 shows S3 RNA sequencing alignments to the wp genomic locus. A zoomed-out genome browser view of the white pupae genomic locus and gene structure (bars on bottom). RNAseq reads were aligned (light grey bars) for the female samples (27023-27025) and the male samples (27026-27028). The inserted tra intron is indicated in dotted box.

[0037] FIGs. 6A-6B show S4 Medfly RNAseq Clustering and Principal Component Analysis. FIG. 7 shows S5 white pupae genotyping for homozy gous IMPERIAL strain generation, where diagrams depict the genotyping strategy' to distinguish the three possible alleles, used at every’ generation from G2 till G7.

[0038] FIG. 8 shows a simplified diagram showing the IMPERIAL strain generation and its underlying mechanism.

[0039] DETAILED DESCRIPTION

[0040] Provided herein are methods of generating a sex-distinguishable insect population or identifying the sex of an insect based on a differential sex-specific phenotype that include (a) delivering a cisgenic nucleic acid molecule into an insect genome, wherein the nucleic acid molecule comprises a sex-specific splicing module, and wherein the nucleic acid molecule is introduced into an endogenous phenotypic gene of the insect; (b) generating the sex-distinguishable insect population, wherein an insect of the sex-distinguishable insect population comprises the cisgenic nucleic acid molecule; and (c) sorting the insect from the sex-distinguishable insect population based on the differential sex-specific expression of the endogenous phenotypic gene, wherein the endogenous phenotypic gene is functionally' expressed in only one sex.

[0041] Also provided herein are cisgenic genetically modified insects comprising a nucleic acid molecule comprising a sex-specific splicing module within an endogenous phenotypic gene of the insect. In some embodiments, the insect is a mosquito or a fruit fly.Atorney Docket No.: 15670-0446WO1

[0042] It is noted that as used in the specification and the appended claims, the singular forms “a”, “an” and “the” refer to one or more (i.e., at least one) of the grammatical object of the article unless the context clearly dictates otherwise. By way of example, “an insect” encompasses one or more insects.

[0043] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0044] Methods of Sex Sorting and Identifying the Sex of an Insect Based on Sex-Specific Gene Expression

[0045] Provided herein are methods of generating a sex-distinguishable insect population that include (a) delivering a cisgenic nucleic acid molecule into an insect genome, wherein the nucleic acid molecule comprises a sex-specific splicing module, and wherein the nucleic acid molecule is introduced into an endogenous phenotypic gene of the insect; (b) generating the sex-distinguishable insect population, wherein an insect of the sex-distinguishable insect population comprises the cisgenic nucleic acid molecule; and (c) sorting the insect from the sex-distinguishable insect population based on the differential sex-specific expression of the endogenous phenofypic gene, wherein the endogenous phenoty pic gene is functionally expressed in only one sex.

[0046] Also provided herein are methods of identifying the sex of an insect based on a differential sex-specific phenotype that include (a) delivering a cisgenic nucleic acid molecule into an insect genome, wherein the nucleic acid molecule comprises a sex-specific splicing module, and wherein the nucleic acid molecule is introduced into an endogenous phenotypic gene of the insect; (b) generating a sex-distinguishable insect population, wherein an insect of the sex-distinguishable insect population comprises the cisgenic nucleic acid molecule; and (c) sorting the insect from the sex-distinguishable insect population based on the differential sex-specific expression of the endogenous phenoty pic gene, wherein the endogenous phenotypic gene is functionally expressed in only one sex.

[0047] Insect(s) can refer to any member of the largest class of the phylum Arthropoda, which is itself the largest of the animal phyla. Insects have segmented bodies, jointed legs, and external skeletons (e.g., exoskeletons). In some embodiments, an insect can include a bedbug, a housefly, a clothes moth, a Japanese beetle, an aphid, a mosquito, a flea, a horsefly, a hornet, a butterfly, or a moth. In some embodiments, the insect can include a tephritid fruitAtorney Docket No.: 15670-0446WO1

[0048] fly selected from Medfly (Ceratitis capitata), Mexfly (Anastrepha ludens), Oriental fruit fly (Bactrocera dorsalis), Olive fruit fly (Bactrocera oleae). Melon fly (Bactrocera cucurbitae), Natal fruit fly (Ceratitis rosa). Cherry fruit fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyroni), Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), Oriental Fruit Fly (Bactrocera dorsalis), West Indian fruit fly (Anastrepha obliqua), the New World screwworm (Cochliomyia hominivorax), the Old World screwworm (Chrysomya bezziana), Australian sheep blowfly / greenbottle fly (Lucilia cuprina), the pink bollworm (Pectinophora gossypiella), the European Gypsy moth (Lymantria dispar), the Navel Orange Worm (Amyelois transitella), the Peach Twig Borer (Anarsia lineatella), the rice stem borer (Tryporyza incertulas), the noctuid moths. Heliothinae, the Japanese beetle (Papilla japonica), White-fringed beetle (Graphognatus spp.). Boll weevil (Anthonomous grandis), the Colorado potato beetle (Leptinotarsa decern lineata), the vine mealybug (Pianococcus ficus), Asian citrus psyllid (diaphorina citri), Spotted wing drosophila (drosophila suzukii), Bluegreen sharpshooter (graphocephala atropunctata), Glassy winged sharpshooter (Flomalodisca vitripennis), Light brown apple moth (Epiphyas postvittana), Bagrada bug (Bagrada hilaris), Brown marmorated stink bug (Halyomorpha halys), Asian Gypsy Moth selected from the group of Lymantria dispar asiatica, Lymantria dispar japonica, Lymantria albescens, Lymantria umbrosa, and Lymantria postalba, Asian longhomed beetle (Anoplophora glabripennis), Coconut Rhinoceros Beetle (Oryctes rhinoceros), Emerald Ash Borer (Agrilus planipennis), European Grapevine Moth (lobesia botrana), European Gypsy- Moth (Lymantria dispar). False Codling Moth (Thaumatotibia leucotreta), fire ants selected from Solenopsis invicta Buren, and S. richteri Forel, Old World Bollworm (Flelicoverpa armigera), Spotted Lantemfly (Lycorma delicatula), Africanized honeybee (apis mellifera scutellata), Fruit and shoot borer (leucinodes orbonalis), com root worm (Diabrotica spp.). Western com rootworm (diabrotica virgifera). Whitefly (bemisia tabaci), Flouse Fly (Musca Domestica), Green Bottle Fly (Lucilia cuprina), Silk Moth (Bombyx mori), Red Scale (Aonidiella aurantia), Dog heartworm (Dirofilaria immitis), Southern pine beetle (Dendroctonus frontalis), Avocado thrip (Thysanoptera Spp.), Botfly selected from Oestridae spp. and Dermatobia hominis), Florse Fly (Tabanus sulcifrons), Flom Fly (Flaematobia irritans), Screwworm Fly selected from Cochliomyia macellaria (C. macellaria), C. hominivorax, C. aldrichi, or C. minima, Tsetse Fly (Glossina spp.), Warble Fly selected from Flypoderma bovis or Hypoderma lineatum, Spotted lantemfly (Lycorma delicatula), Khapra beetle (Trogoderma granarium). Honeybee mite (Varroa destmctor), Termites (Coptotermes formosanus), Hemlock woolly adelgid (Adelges tsugae). Walnut twig beetle (PityophthorusAtorney Docket No.: 15670-0446WO1

[0049] juglandis), European wood wasp (Sirex noctilio), Pink-spoted bollworm (pectinophora scutigera), Two spoted spider mite (Tertanychus urticae), Diamondback moth (plutella xylostella), Taro caterpillar (spodoptera litura), Red flour beetle (tribolium castaneum), Green peach aphid (Myzus persicae), Coton Aphid (aphis gossypii), Brown planthopper (nilaparvata lugens), Beet armyworm (spodotera exigua), Western flower thrips (frankliniella occidentalis), Codling moth (cydia pomonella), Cowpea weevil (callosobruchus maculatus), Pea aphid (acyrthosiphon pisum), Tomato leafminer (tuta absoluta), Onion thrips (thrips tabaci), or Coton bollworm (Helicoverpa armigera). In some embodiments, the insect is Ceratitis capitata. In some embodiments, an insect can be a mosquito. In some embodiments, the mosquito can include Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus (Aedes triseriatus). Anopheles stephensi, Anopheles albimanus, Anopheles gambiae. Anopheles quadrimaculatus, Anopheles freebomi, Culex species, or Culiseta melanura.

[0050] Sex-specific splicing modules

[0051] In some embodiments, any one of the methods described herein can exploit sexspecific expression, via sex-specific alternative splicing (SSAS), of a phenotypic gene. In some embodiments, any one of the methods described herein can exploit female specific expression via sex-specific alternative splicing (SSAS) of the phenotypic gene. In some embodiments, any one of the methods described herein can exploit male specific expression via sex-specific alternative splicing (SSAS) of the phenotypic gene. In some embodiments, sex-sorting of an insect and / or identifying a sex of an insect can identify the insect as a male. In some embodiments, sex-sorting of an insect and / or identifying a sex of an insect can identity' the insect as a female.

[0052] In some embodiments, splicing refers to an essential and highly regulated gene expression mechanism whereby some sequences (e.g., introns) are excised from pre-mRNAs, and others (e.g., exons) are presen' ed and consecutively joined to produce mature mRNAs. As a result, the splicing machinery may process pre-mRNAs (i.e., recognize intron and exon sequences) in different alternative manners (“alternative splicing”) to produce different mRNA species (alternatively spliced mRNA species, or splicing isoforms) from a single gene. In some embodiments, a sex-specific splicing module can include a polynucleotide construct that, when introduced into an insect, undergoes differential splicing (e.g., sexspecific) and thus creates a different transcript in females than males. In some embodiments,Atorney Docket No.: 15670-0446WO1

[0053] the sex-specific splicing module provides female specific protein expression. In some embodiments, the sex-specific splicing module provides male specific protein expression.

[0054] In some embodiments, the sex-specific splicing module allows sex-specific alternative splicing of a phenotypic gene, wherein a phenotypic gene refers to a gene that manifests as an observable trait or phenotype, such as color, wing shape, body size, behavior, body morphology, antennae shape, leg structure, or abdominal shapes. In some embodiments, a phenotypic gene in an insect comprises a gene whose mutation causes the insect pupae to be a different color from the pupae color of a wild-ty pe insect. In some embodiments, the phenotypic gene comprises a white pupae (wp) gene. In some embodiments, the sex-specific splicing module activates the expression of the phenotypic gene. In some embodiments, the sex-specific splicing module disrupts the expression of the phenotypic gene.

[0055] In some embodiments, the sex-distinguishable insect population is generated by cisgenic genetic modification. A cisgenic genetic modification can refer to the genetic modification of an insect using genes from the same species, wherein the genes are controlled by their native promoter. In some embodiments, a sex-specific splicing module comprises an endogenous sex-specific intromc sequence. In some embodiments, the endogenous sexspecific intronic sequence includes an intron that is alternatively spliced in males compared to females. In some embodiments, the endogenous sex-specific intronic sequence comprises an endogenous transformer (Ira) intron.

[0056] In some embodiments, any one of the methods described herein comprises sex-sorting a plurality of insects based on sex-specific gene expression. Sex-sorting can refer to sorting and separating an insect from a plurality of insects from the same species (e.g., Aedes aegypti, Drosophila melanogaster. Drosophila suzukii, Ceratitis capitata, or Anastrepha ludens), thereby soring the plurality of insects into two groups (e.g., male and female). In some embodiments, an insect of a plurality of insects can be identified as a male insect. In some embodiments, an insect of a plurality of insects can be identified as a female insect. In some embodiments, an insect is sorted as male based on the expression of the phenotypic gene. In some embodiments, the insect is sorted as female based on the expression of the phenotypic gene. In some embodiments, an insect can be removed from the plurality of insects once it is identified as a male insect. In some embodiments, an insect can be removed from the plurality of insects once it is identified as a female insect. In some embodiments, the differential sex-specific expression of the endogenous phenotypic gene comprises molecular detection, behavioral phenotype, viability differences, or size or timing differences.Atorney Docket No.: 15670-0446WO1

[0057] In some embodiments, sex-sorting can include sorting a plurality of insects according to a difference in pupae color between female and male insects. In some embodiments, a pupae of a female insect can be a brown color, while a pupae of a male insect can be a white color. In some embodiments, the insect is sex-sorted by automated machine-vision sorting, fluorescence-based sorting, or mechanical sex-separation enhanced by pupae color.

[0058] In some embodiments, sex-sorting can include sorting a plurality of insects according to a difference in size between female and male insects. In some embodiments, the size of a female insect can be different from a male insect at the same developmental stage. In some embodiments, the insect is sex-sorted by automated machine-vision sorting, fluorescencebased sorting, or mechanical sex-separation based on developmental size.

[0059] In some embodiments, any one of the methods described herein can enable sexsorting of an insect during early larval development stage, the pupal developmental stage, or the adult stage. In some embodiments, any one of the methods described herein can enable sex-sorting of an insect as a pupae.

[0060] Delivery of nucleic acid molecule

[0061] In some embodiments, any one of the methods described herein includes delivery (e.g., gene delivery, gene transfer) of a nucleic acid molecule (e.g., polynucleotide) into an insect genome, wherein the delivery includes introducing the nucleic acid molecule into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (e.g., viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (e.g., electroporation, “gene gun” delivery' and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.

[0062] In some embodiments of any of the methods described herein, introducing a nucleic acid molecule into an insect genome is facilitated by a gene-editing agent, transposase-mediated insertion, homology-directed repair (HDR), non-homologous end-joining (NHEJ)Atorney Docket No.: 15670-0446WO1

[0063] repair, or recombinase-mediated cassette exchange. In some embodiments, the nucleic acid molecule is delivered into the insect at the embryo stage of development of the insect.

[0064] A gene-editing agent can refer to an agent that allows for changing the DNA or RNA (e.g., mRNA) in the genome. In some embodiments, gene-editing can include insertion, deletion, modification, or replacement of the DNA or RNA. In some embodiments, a geneediting agent can include a nuclease-based gene editing platform. In some embodiments, a gene-editing agent can include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), engineered meganucleases, or a clustered regularly interspaced short palindromic repeats (CRISPR) system. In some embodiments, a gene-editing agent can include RNA base editors (e g., ADAR) or DNA base editors (e.g., target- AID base editor and dddA-derived cytosine base editor). In some embodiments, a gene-editing agent can include a CRISPR-associated transposase. In some embodiments, a gene-editing agent can include RNA interference (e.g., short hairpin RNA (shRNA), small interfering RNA (siRNA), antisense oligonucleotide (ASO), or microRNA mimics). In some embodiments, the gene-editing agent can include an RNA guide gene targeting system. In some embodiments, the gene-editing agent can include CRISPR components. For example, in some embodiments, CRISPR components can include, but are not limited to, a guide RNA and a CRISPR-associated endonuclease (Cas protein). As used herein, a “CRISPR-associated endonuclease'’ or “CRISPR-associated protein’" can refer to an enzyme or protein that uses CRISPR sequences as a guide to recognize and cleave specific nucleic acid strands that are complementary to the CRISPR sequence. In some embodiments, a gene-editing agent can include a CRISPR-associated protein. In some embodiments, the gene-editing agent can be a Cas9 endonuclease that makes a double-stranded break in a target DNA sequence. In some embodiments, the gene-editing agent can include a Cas9 protein. In some embodiments, the gene-editing agent can be a Casl2 (e.g., Casl2a) nuclease that also makes a double-stranded break in a target DNA sequence. In some embodiments, a gene-editing agent can irreversibly knock out the target gene via double strand breaks. In some embodiments, a gene-editing agent that irreversibly knocks out the target gene can be a Cas9 nuclease which targets RNA. In some embodiments, a gene-editing agent can transiently reduce the expression of a target gene. In some embodiments, a gene-editing agent that transiently reduces the expression of a target gene can include a Cas protein that targets RNA, including but not limited to Cas9, Cas 13, Cmr, and Csm systems. In some embodiments, the gene-editing agent is selected from the group consisting of a CRISPR / Cas9 system, a CRISPR / Casl2 system, a Cas 13 system, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs),Atorney Docket No.: 15670-0446WO1

[0065] homology-directed repair, non-homologous end-joining (NHEJ) repair, and recombinases. In some embodiments, the gene-editing agent comprises CRISPR / Cas9 components.

[0066] In some embodiments of any of the methods described herein, introducing a nucleic acid molecule into an insect genome is facilitated by transposase-mediated insertion.

[0067] Transposase-mediated insertion refers to a biological process wherein an enz me (e.g., transposase) cuts a mobile piece of DNA, a transposon, from one location in the genome and inserts it into a new target site, often recognizing specific inverted repeats at the transposon's ends to excise and insert it, leading to genetic variation or functional changes. In some embodiments of any of the methods described herein, introducing a nucleic acid molecule into an insect genome is facilitated by homologous recombination (HR). In some embodiments, HR can be a conservative process that uses a homologous template (e.g., the sister chromatid in S or G2 phases of the cell cycle, the homologous chromosome), to repair the damaged section of DNA. In some embodiments of any of the methods described herein, introducing a nucleic acid molecule into an insect genome is facilitated by non-homologous end joining (NHEJ). In some embodiments, end joining pathways, including NHEJ, can be error prone and often mutagenic. In some embodiments, NHEJ involves ligation of ends of the broken DNA, often adding or deleting nucleotides prior to ligation, which results in deletions and frameshifts.

[0068] Cisgenic Genetically Modified Insect

[0069] Provided herein are cisgenic genetically modified insects that include a nucleic acid molecule comprising a sex-specific splicing module within a phenotypic gene of the insect. In some embodiments, the cisgenic genetically modified insect is a mosquito or a fruit fly. In some embodiments, the cisgenic genetically modified insect is a tephritid fruit fly selected from Medfly (Ceratitis capitata), Mexfly (Anastrepha ludens), Oriental fruit fly (Bactrocera dorsalis). Olive fruit fly (Bactrocera oleae), Melon fly (Bactrocera cucurbitae), Natal fruit fly (Ceratitis rosa). Cherry fruit fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyrom). Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), Oriental Fruit Fly (Bactrocera dorsalis), West Indian fruit fly (Anastrepha obliqua), the New World screwworm (Cochliomyia hominivorax), the Old World screwworm (Chrysomya bezziana), Australian sheep blowfly / greenbottle fly (Lucilia cuprina), the pink bollworm (Pectinophora gossypiella), the European Gypsy moth (Lymantria dispar), the Navel Orange Worm (Amyelois transitella), the Peach Twig Borer (Anarsia lineatella), the rice stem borerAtorney Docket No.: 15670-0446WO1

[0070] (Tryporyza incertulas), the noctuid moths, Heliothinae, the Japanese beetle (Papilla japonica), White-fringed beetle (Graphognatus spp.), Boll weevil (Anthonomous grandis), the Colorado potato beetle (Leptinotarsa decern lineata), the vine mealybug (Pianococcus ficus), Asian citrus psyllid (diaphorina citri), Spotted wing drosophila (drosophila suzukii). Bluegreen sharpshooter (graphocephala atropunctata), Glassy winged sharpshooter (Flomalodisca vitripennis), Light brown apple moth (Epiphy as postvittana), Bagrada bug (Bagrada hilaris), Brown marmorated stink bug (Halyomorpha halys), Asian Gypsy Moth selected from the group of Lymantria dispar asiatica, Lymantria dispar japonica, Lymantria albescens, Lymantria umbrosa, and Lymantria postalba, Asian longhomed beetle (Anoplophora glabripennis), Coconut Rhinoceros Beetle (Oryctes rhinoceros), Emerald Ash Borer (Agrilus planipennis). European Grapevine Moth (lobesia botrana), European Gypsy Moth (Lymantria dispar), False Codling Moth (Thaumatotibia leucotreta), fire ants selected from Solenopsis invicta Buren, and S. richteri Forel, Old World Bollworm (Flelicoverpa armigera), Spotted Lantemfly (Lycorma delicatula), Africanized honeybee (apis mellifera scutellata), Fruit and shoot borer (leucinodes orbonalis), com root worm (Diabrotica spp ). Western com rootworm (diabrotica virgifera), Whitefly (bemisia tabaci), Flouse Fly (Musca Domestica), Green Bottle Fly (Lucilia cuprina). Silk Moth (Bombyx mori), Red Scale (Aonidiella aurantia), Dog heartworm (Dirofilaria immitis), Southern pine beetle (Dendroctonus frontalis), Avocado thrip (Thysanoptera Spp.), Botfly selected from Oestridae spp. and Dermatobia hominis), Florse Fly (Tabanus sulcifrons), Flom Fly (Flaematobia irritans). Screwworm Fly selected from Cochliomyia macellaria (C. macellaria), C. hominivorax, C. aldrichi, or C. minima, Tsetse Fly (Glossina spp.), Warble Fly selected from Flypoderma bovis or Hypoderma lineatum, Spotted lantemfly (Lycorma delicatula), Khapra beetle (Trogoderma granarium), Honeybee mite (Varroa destructor), Termites (Coptotermes formosanus). Hemlock woolly adelgid (Adelges tsugae), Walnut twig beetle (Pityophthorus juglandis), European wood wasp (Sirex noctilio), Pink-spotted bollworm (pectinophora scutigera), Two spotted spider mite (Tertanychus urticae), Diamondback moth (plutella xylostella), Taro caterpillar (spodoptera litura), Red flour beetle (tribolium castaneum), Green peach aphid (Myzus persicae), Cotton Aphid (aphis gossypii), Brown planthopper (nilaparvata lugens), Beet armyworm (spodotera exigua), Western flower thrips (frankliniella occidentalis). Codling moth (cydia pomonella), Cowpea weevil (callosobruchus maculatus), Pea aphid (acyrthosiphon pisum), Tomato leafminer (tuta absoluta), Onion thrips (thrips tabaci), or Cotton bollworm (Helicoverpa armigera). In some embodiments, the cisgenic genetically modified insect is a Ceratitis capitata (Mediterranean fruit fly).Atorney Docket No.: 15670-0446WO1

[0071] In some embodiments, the cisgenic genetically modified insect is a mosquito selected from the group consisting of Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus (Aedes triseriatus). Anopheles stephensi, Anopheles albimanus, Anopheles gambiae.

[0072] Anopheles quadrimaculatus, Anopheles freebomi, Culex species, and Culiseta melanura.

[0073] EXAMPLES

[0074] The disclosure is further described in the following examples, which do not limit the scope of the disclosure.

[0075] Materials & Methods

[0076] Plasmid design and construction

[0077] Gibson enzymatic assembly was used to build the 1167A plasmid, which contains C. capitata MFS transporter Exon 3 (LOCI 01451947). the tra intron and Opie2-DsRed outside the homology arms. A pre-existing plasmid containing piggyBac flanks, with an Opie2 promoter regulating DsRed, was linearised by Ndel and Kpnl to clone 1167A. The MFS transporter Exon3 was amplified into two fragments from C. capitata genomic DNA using primer pairs 1167A.C1F and c2R, as well as 1167A.c5F and c6R (Table 1). The tra intron was amplified from 795H1 (Addgene #205482) using primer pair 1167A.c3F and c4R, then inserted inside of the MFS exon 3 coding sequence (Table 1).

[0078] [Table 1] Primer Summary

[0079]

[0080] Atorney Docket No.: 15670-0446WO1

[0081]

[0082] C. capitata maintenance

[0083] All fly stains were reared under standard lab conditions. A carrot-based diet was provided for larval development and a 1 : 1 yeast: glucose mix was given to adult flies. The Benakeion wild- t pe strain was supplied by the Saccone Lab (University of Naples ‘'Federico II”), whilst irradiation-generated white pupae - / - (wp- / -) and VIENNA 8 D53- strains w ere obtained from the FAO / IAEA Centre of Nuclear Techniques in Food and Agriculture (Seibersdorf, Austria).

[0084] C. capitata germline transformation

[0085] Microinjections of the 1167A plasmid were performed into embryos of the wild-type Benakeion strain. The plasmid (250 ngZj.il ) was injected alongside a pre-assembled ribonucleoprotein (RNP) complex of Cas9 protein (200 ng / pl) (PNA Bio) and presynthesized gRNA-wp (100 ng / pl) (Synthego).

[0086]

[0087] The cross scheme for G0-G3 line generation is summarized in FIGs. 1A-1B. The injected GOs were reciprocally crossed to the characterised irradiation-generated wp- / - strain. The resulting G1 progeny was separated by pupal colour, and all males emerging from whi te pupae w ere backcrossed in pools to the females from the irradiation-generated wp- / - strain. At G2, the brown pupae were isolated, and all 10 female adults from the same parental G1 cross were collectively crossed to five sibling white-pupaed males. After mating, genomic DNA (gDNA) was individually extracted from G2 brown-pupaed females using an altered phenol-chloroform protocol. The knock-in site was amplified using Phusion High-Fidelity PCR Master Mix with HF Buffer (New England Biolabs®) with genome-specific 1167A_F and 1167A_R primers (Table 1) designed in Geneious Prime 2023.1.2. To confirm the initialAtorney Docket No.: 15670-0446WO1

[0088] integration, the PCR products were purified via Monarch® PCR & DNA Cleanup Kit (New England Biolabs®) and analysed via Oxford Nanopore sequencing (Full Circle Labs).

[0089] Over the course of the following 4 generations (G3-G6) multiple sibling crosses were performed in parallel and the parental genotypes were verified. Hereby three possible alleles were differentiated: 1) the irradiation-generated wp- / -, 2) the intron-less wp- / - with indels and 3) the tra intron knock-in (FIG. 7). For this, a multiplex 3-primer PCR was designed using a forward (1167 A_F) binding upstream of the integration site, a reverse (1167A_R) downstream of the integration site and a second reverse (8kb_B_R) binding to the irradiationgenerated wp- / - inserted sequence (FIG. 7).

[0090] Verification of genome integration site in the homozygous strain

[0091] To verify the insertion site in the wp+ / +IMPERIAL strain, Oxford Nanopore genomic DNA sequencing was conducted. Genomic DNA was extracted from four knock-in adult males and four knock-in adult females using the Blood & Cell Culture DNA Midi Kit (Qiagen).

[0092] PCR tra intron splicing confirmation

[0093] In parallel, single males and females were separately collected for gDNA and RNA extractions. gDNA was extracted as detailed above. RNA as extracted via an adapted TRIzol® (Ambion)-chloroform-based protocol. CDNA was synthesised from total RNA using Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase (ThermoFisher) according to the instructions provided by the manufacturer. Phusion High-Fidelity PCR Master Mix with HF Buffer (New England Biolabs®) w as used for PCR amplification from gDNA and cDNA templates using the 1167_F_V1 splicing and 1167A_R primer pair (Table 1). Bands amplified from cDNA of the IMPERIAL strain were purified using a Monarch® DNA Gel Extraction Kit (New- England Biolabs®) and Sanger sequenced (Genewiz Inc.). Male cDNA PCR reaction was additionally subjected to Sanger sequencing post-PCR product cloning using the StrataClone PCR Cloning Kit (Agilent) whereby different isoforms were isolated.

[0094] RNA sequencing

[0095] To confirm the sex-specific expression of the wp gene, Illumina RNA sequencing was performed. Total RNA was extracted from mature wpKIadult males and females from the IMPERIAL strain in three biological replicates (six samples) using the miRNeasyAtorney Docket No.: 15670-0446WO1

[0096] Tissue / Cells Advanced Mini Kit (Qiagen), following the manufacturer's protocol. Genomic DNA was removed using the gDNA eliminator column included with the kit. RNA integrity was tested with the RNA 6000 Pico Kit for Bioanalyzer (Agilent Technologies).

[0097] Male (ID# 27026-27028) and female (ID# 27023-27025) libraries with three replicates each were sequenced to approximate depth of 20M paired end reads. The reads were aligned with STAR (github.com / alexdobin / STAR) to the EGII-3.2.1 genome assembly (www.ncbi.nlm.nih.gov / datasets / genome / GCA_905071925.1 / ), into which traF intron was inserted at the white pupae locus (GCA_905071925.1_EGII-3.2. l_genomic.traF-intron.fna). To generate a more complete annotation fde the Ccap_2.1 annotations were transferred (www.ncbi.nlm.nih.gov / datasets / genome / GCA 000347755.4 / ) to the EGII-3.2.1 genome by aligning Ccap_2.1 transcript sequences with BLAT and parsing the alignments to generate a GTF file, which was used for all subsequent analysis steps. Gene abundances were quantified with featureCounts (subread.sourceforge.net / featureCounts.html), count data were converted to TPM and FPKM values and combined using Perl scripts. Gene annotations were downloaded from EnsemblMetazoa using the BioMart tool (metazoa.ensembl.org / Ceratitis_capitata_gca000347755v4 / Info / Index) and added to the quantification data. TPM values were used to perform PCA and clustering analyses in R to identify possible sample outliers. Replicates for each sex clustered together as expected and displayed high correlations between each other without obvious outliers (FIGs. 6A-6B). To visualize splicing of the tra intron within the white pupae locus (LOCI 01451947), BAM files produced by STAR were imported into IGV (igv.org / doc / desktop / ).

[0098] Stability assay

[0099] From G3 until G7 all pupae were separated by colour and corresponding adults were scored by sex. When the homozygosity of the IMPERIAL strain was verified at G7 (FO), alongside the VIENNA 8 D53- strain, for 5 consecutive generations (F2-F6) eggs were collected five days after eclosion and raised under regular conditions until pupal stage of development. Brown and white pupae were then separated, and the sex of all adults from each pool was recorded.

[0100] Egg-adult survival assay

[0101] Sibling crosses of 10 males and 20 females were set up in triplicates simultaneously for the IMPERIAL, VIENNA 8 D53-, and wild-type Benakeion strains. Egg numbers and theirAtorney Docket No.: 15670-0446WO1

[0102] hatching rates were determined. Specifically, five days after eclosion, all eggs laid within a 5-hour period were collected and unhatched eggs were counted twice four days apart. Pupal and adult recovery rates were determined thereafter.

[0103] Adult longevity assay

[0104] Age-matched adults from the wild-ty pe Benakeion, IMPERIAL and VIENNA 8 D53- strains were separated by sex upon eclosion and placed into cages of 10 individuals. Three male and three female replicates were set up simultaneously for each strain; and maintained under standard conditions thereafter. Daily, dead flies were counted and removed from the cages for 30 consecutive days.

[0105] Mating-preference assay

[0106] 90 females from the irradiation-generated wp- / - strain were simultaneously placed together with 15 males from each of the Benakeion, IMPERIAL and VIENNA 8 D53- strains for a total of a 1:2 male: female ratio. The experiment was repeated 3 times. The flies were left to mate for 4 full days, after which females were separated into individual small cages. Upon oviposition, eggs from all females which oviposited were separately collected and reared normally until pupation. In the cases where white and brow n pupae were present, they were separated by colour. Adults eclosing from both mixed and brown pupae-only collections were screened by sex.

[0107] Eclosion assay

[0108] 24-hour egg collections were made from sibling crosses of 10 males and 20 females of the IMPERIAL, VIENNA 8 D53-, and wild-type Benakeion strains, set up in parallel triplicates. The offspring were reared under normal conditions until pupation, whereby IMPERIAL and VIENNA 8 pupae w ere sorted by colour. The eclosing adults were therein scored by sex every day.

[0109] Example 1 - Cisgenic genetic sexing strains

[0110] To generate minimal genetic modifications wherein both donor and recipient are derived from the same species, referred to herein as cisgenic GSS (CGSS), the medfly was used as the model system to engineer a non-transgenic CGSS without exogenous elements. Sex-specific expression of the white pupae (wp) selectable marker was achieved through sexspecific splicing of the tra intron. This was attained through a homology-directed repairAtorney Docket No.: 15670-0446WO1

[0111] (HDR)-dependent knock-in of the tra intron seamlessly into the wp locus of wild-ty pe Benakeion medfly embry os, feasible due to recent success of CRISPR / Cas9-mediated HDR in the species.

[0112] Collectively, using the intron of tra, a master gene of tephritid female sex determination, results in extremely robust desired phenoty pes, rendering it suitable for further use in CGSSs which have not been developed previously. The knock-in construct (1167 A) was engineered using the endogenous transformer (tra) intron, which was placed between the homology arms of each approximately 700 bp in length. The anticipated outcome was female-specific wp gene expression, whereby males and females harbouring two copies of the / ra-contaming wp gene would have white and brown pupae phenoty pes accordingly which are visible by eye (FIG. 1A). As the wp mutation is recessive in nature, the knock-in strain, hereupon named '‘IMPERIAL”, was isolated using backcrosses to the irradiation-generated white pupae knock-out (wp- -) strain at GO and G1 (FIG. IB). Successful knock-in of the tra intron (wjfI+ / ~ in all obtained brown-pupaed G2 females was confirmed using amplicon sequencing of the integration site. A homozygous wpi:!line was established at G7 (F0) after crossing sibling white-pupaed males with brown-pupaed females, genotyping all parents and screening the whole progeny at every intermediate generation. To verify the integration in the f:' IMPERIAL strain, genomic DNA from CGSS females was sequenced. The sequencing revealed reads with the expected 1345 bp CRISPR-HDR insertion indicating a seamless intragenic insertion of the tra intron into the wp gene (FIG. 3).

[0113] Example 2 - Sex-sorting suitability of the CGSS

[0114] To verify the sex-sorting suitability' of the strain, phenotypic pupae colour stability and sex ratios of the IMPERIAL strain were examined alongside the existing VIENNA 8 GSS, in which males and females emerge from brown and white pupae accordingly. For five consecutive generations (F2-F6), pupae and adult phenoty pes were recorded for IMPERIAL (pupae n = 4147; adult n = 4074) and VIENNA 8 (pupae n = 1386; adult n = 1227) strains in parallel, maintained under the same conditions. As anticipated, in the vpf IMPERIAL strain all females emerged from brown pupae and all males emerged from white pupae (FIG.

[0115] 2A). The reverse phenotypes were universally^ observed amongst individuals from the VIENNA 8 strain. Altogether, the proportion of adult females in the IMPERIAL strain (mean = 49.8%) exceeded that of the VIENNA 8 strain (mean = 37.4%). Whilst populations within every generation of the IMPERIAL strain adhered to the expected 1:1 male: female sex ratio (chi-square goodness of fit tests), the VIENNA 8 did not at F2, F4 and F5 (FIG. 2A). In sumAtorney Docket No.: 15670-0446WO1

[0116] these results confirm the phenoty pic stability' of the IMPERIAL strain which, dissimilarly to VIENNA 8, consistently confines to the expected 1:1 male: female sex ratio.

[0117] To better understand differences in the tra intron-dependent wp splicing in males and females of theWpm+ / + IMPERIAL strain, reverse-transcription PCR (RT-PCR) was performed on genomic and complementary7DNA (cDNA) templates from adult flies (FIGs.

[0118] 4A-4B). A single female band was amplified from the cDNA template, corresponding to a transcript with the fully spliced-out tra intron, which was confirmed by sequencing. Male cDNA banding entirely consisted of larger fragments from which two unique male isoforms with premature stop codons were isolated via clonal sequencing, both containing sequences of the two male-specific exons. These results are suggestive of functional White pupae protein production in females and ablation of its translation in males. Further investigation into wp splicing of the IMPERIAL strain was conducted using RNAseq. As expected, all three female libraries had multiple reads that spanned the junction splicing the tra intron. In contrast, the three male libraries had no such reads indicating that the intron is spliced in females and not in males (FIG. 5). As expected, the clustering and principal component analyses indicated a close relationship of samples by sex (FIGs. 6A-6B).

[0119] Example 3 - Suitability for larger-scale employment

[0120] The IMPERIAL sexing strain was further compared to its parental wild-type Benakeion and VIENNA 8 strains in terms of general fitness, and thus suitability for larger-scale employment. First, a standard egg-adult survival assay utilising sibling crosses was conducted whereby rates of egg laying, egg hatching, hatched lan ae-pupae recovery7, pupae-adult recovery, and total egg-adult recovery were assessed (FIG. 2A). Kruskal-Wallis and sequential Dunn’s tests were performed as means of statistical analysis. Notably, egg production within the measured time period was significantly elevated in the IMPERIAL strain compared to both wild-type and VIENNA 8 strains (p < 0.05*). Hatching rate in VIENNA 8 w as significantly lower than in wild-ty pe (p = 0.0036**), although insignificant reductions in the IMPERIAL strain were also observed (p = 0.0899). The highest larval-pupal survival was recorded in the IMPERIAL strain, which was significantly raised compared to VIENNA 8 (p = 0.0036**). In line with the earlier phenotypic stability experiments, the pupal-adult recovery7rates were significantly low er in the VIENNA 8 strain, compared to both wild-type and IMPERIAL strains (p < 0.05*). Overall egg-adult survival was significantly reduced in VIENNA 8 when assessed against wild-type (p = 0.0127*) and IMPERIAL (p = 0.0368*) strains alike. Egg-adult survival in wild-type and IMPERIALAttorney Docket No.: 15670-0446WO1

[0121] strains, however, was statistically similar (p = 0.3274), indicative of good fitness in the IMPERIAL strain.

[0122] Example 4 - Adult longevity in males and females

[0123] 5 Adult longevity was explored with virgin males and females restricted to separate husbandry under regular lab conditions (FIG.2C). Highest survival was observed in wildtype females, whilst the shortest longevity belonged to VIENNA 8 females. Pairwise comparisons via log-rank tests were performed between strains, and the combinations of stain and sex (Tables 2 and 3). Altogether, VIENNA 8 had significant reductions in longevity

[0124] 10 compared to both wild-type (p = 0.0022**) and IMPERIAL (p = 0.0048**) strains. Wildtype Benakeion and IMPERIAL strains with the same genetic background, on the other hand, did not have a significant difference between one another (p = 0.3920). Amongst females, significant differences were recorded for VIENNA 8 against both wild-type (p < 0.0001****) and IMPERIAL (p < 0.0001****) strains, while male comparisons between strains were

[0125] 15 statistically insignificant. These results are suggestive of comparable longevity of the males and importantly females from the IMPERIAL strain with wild-type.

[0126] [Table 2] Statistical analysis of adult longevity by strain

[0127]

[0128] 20 [Table 3] Statistical analysis of adult longevity by strain and sex

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[0134]

[0135] Attorney Docket No.: 15670-0446WO1

[0136] Example 5 - Delay in development in males and females

[0137] A delay in white-pupaed female development has been documented in the VIENNA 8 strain (40). To determine whether similar issues occur in the white-pupaed males of the IMPERIAL strain, we compared pupal eclosion times from age-matched egg collections by sex. The development times were significantly different by strain and sex (nested ANOVA, F = 178.6, p < 0.0001****) (FIG. 2D). The time for wild-type males (mean = 18.60 days) and females (mean = 18.75 days) to reach adulthood were statistically similar to IMPERIAL males (mean = 18.51 days) and females (mean = 18.59 days) (Table 4). Flies from the VIENNA 8 strain were significantly slower to eclose, with means of 20.11 and 24.00 days for males and females accordingly. Whilst there may be differences in acclimation of the VIENNA 8 to a newer laboratory environment in comparison to the other two strains, there was a significant difference between VIENNA 8 males and females (p < 0.0001****), which is absent in both wild-type (p = 0.94854) and IMPERIAL (p = 0.99781) strains. The observed trends strongly indicate that the IMPERIAL strain does not experience a developmental discrepancy between sexes which is present in the current VIENNA 8 GSS.

[0138] [Table 4] Statistical analysis of pupal eclosion times by strain and sex

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[0145] Example 6 - Mating preferences of females

[0146] The mating preferences of females towards the IMPERIAL, Benakeion and VIENNA 8 strains were assessed through simultaneous allowing of mating between males from all three strains with females from the irradiation-generated wp-,'- strain. Due to the recessiveAttorney Docket No.: 15670-0446WO1

[0147] nature of the wp mutation, the father(s) were easily ‘revealed' through pupal colour and corresponding adult phenotype screening (Table 5). The most common parentage belonged to wild- type male(s) (36.93%), followed by equal parentage by IMPERIAL male(s) (28.41%) and VIENNA 8 male(s) (28.41%). We also observed progeny with mixed strain paternity at a low' frequency (6.25%). This data indicates that the pupal colour of the IMPERIAL males does not disadvantage their mating success in respect to brown-pupaed VIENNA 8 males.

[0148] [Table 5] Mating preference of females tow ards wild-type, IMPERIAL and VIENNA 8 strains

[0149]

Claims

Attorney Docket No.: 15670-0446WO1WHAT IS CLAIMED IS:

1. A method of generating a sex-distinguishable insect population, the method comprising:(a) delivering a cisgenic nucleic acid molecule into an insect genome, wherein the nucleic acid molecule comprises a sex-specific splicing module, and wherein the nucleic acid molecule is introduced into an endogenous phenotypic gene of the insect;(b) generating the sex-distinguishable insect population, wherein an insect of the sex-distinguishable insect population comprises the cisgenic nucleic acid molecule; and(c) sorting the insect from the sex-distinguishable insect population based on the differential sex-specific expression of the endogenous phenotypic gene,wherein the endogenous phenotypic gene is functionally expressed in only one sex.

2. The method of claim 1, wherein the sex-specific splicing module activates the expression of the endogenous phenotypic gene.

3. The method of claim 1, wherein the sex-specific splicing module disrupts the expression of the endogenous phenotypic gene.

4. The method of any one of claims 1-3, wherein the sex-specific splicing module comprises an endogenous sex-specific intronic sequence.

5. The method of claim 4, wherein the endogenous sex-specific intronic sequence comprises an endogenous transformer fra) intron.

6. The method of any one of claims 1-5, wherein the endogenous phenotypic gene comprises awhite pupae (wp) gene.

7. The method of any one of claims 1-6, wherein the cisgenic nucleic acid molecule is introduced into the endogenous phenotypic gene of the insect by using a gene-editing agent, transposase-mediated insertion, HDR replacement, or recombinase-mediated cassette exchange.Attorney Docket No.: 15670-0446WO18. The method of claim 7, wherein the gene-editing agent is selected from the group consisting of a CRISPR / Cas9 system, a CRISPR / Casl2 system, a Casl3 system, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), homology-directed repair, non-homologous end-joining (NHEJ) repair, and recombinases.

9. The method of claim 7, wherein the gene-editing agent comprises CRISPR / Cas9 components.

10. The method of any one of claims 1-9, wherein the cisgenic nucleic acid molecule is delivered into the insect at the embry o stage of development of the insect.

11. The method of any one of claims 1-10, wherein the insect is sorted as male based on the expression of the endogenous phenotypic gene.

12. The method of any one of claims 1-10, wherein the insect is sorted as female based on the expression of the endogenous phenotypic gene.

13. The method of any one of claims 1-12, wherein the differential sex-specific expression of the endogenous phenotypic gene comprises molecular detection, behavioral phenotype, viability differences, or size or timing differences.

14. The method of any one of claims 1-13, wherein the insect is sorted by automated machine-vision sorting, fluorescence-based sorting, or mechanical sex-separation enhanced by phenotype.

15. The method of any one of claims 1-14, wherein the insect is sorted at the larval stage, the pupal developmental stage, or the adult stage.

16. The method of any one of claims 1-15, wherein the insect is a mosquito or a fruit fly.

17. The method of claim 16, wherein the insect is a tephritid fruit fly selected from Medfly (Ceratitis capitata), Mexfly (Anastrepha ludens), Oriental fruit fly (Bactrocera dorsalis). Olive fruit fly (Bactrocera oleae), Melon fly (Bactrocera cucurbitae). Natal fruit fly (Ceratitis rosa). Cherry fruit fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyroni),Attorney Docket No.: 15670-0446WO1Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), Oriental Fruit Fly (Bactrocera dorsalis), West Indian fruit fly (Anastrepha obliqua), the New World screwworm (Cochliomyia hominivorax), the Old World screwworm (Chrysomya bezziana), Australian sheep blowfly / greenbottle fly (Lucilia cuprina), the pink bollworm (Pectinophora gossypiella), the European Gypsy moth (Lymantria dispar), the Navel Orange Worm (Amyelois transitella), the Peach Twig Borer (Anarsia lineatella). the rice stem borer (Tryporyza incertulas), the noctuid moths, Heliothinae, the Japanese beetle (Papilla japonica), White-fringed beetle (Graphognatus spp.), Boll weevil (Anthonomous grandis), the Colorado potato beetle (Leptinotarsa decern lineata), the vine mealybug (Pianococcus ficus), Asian citrus psyllid (diaphorina citri), Spotted wing drosophila (drosophila suzukii), Bluegreen sharpshooter (graphocephala atropunctata), Glassy winged sharpshooter (Flomalodisca vitripennis). Light brown apple moth (Epiphyas postvittana), Bagrada bug (Bagrada hilaris), Brown marmorated stink bug (Halyomorpha halys), Asian Gypsy Moth selected from the group of Lymantria dispar asiatica, Lymantria dispar japonica, Lymantria albescens, Lymantria umbrosa, and Lymantria postalba, Asian longhomed beetle (Anoplophora glabripennis), Coconut Rhinoceros Beetle (Oryctes rhinoceros), Emerald Ash Borer (Agrilus planipennis), European Grapevine Moth (lobesia botrana), European Gypsy Moth (Lymantria dispar), False Codling Moth (Thaumatotibia leucotreta), fire ants selected from Solenopsis invicta Buren, and S. richteri Forel, Old World Bollworm (Flelicoverpa armigera), Spotted Lantemfly (Lycorma delicatula), Africanized honeybee (apis mellifera scutellata), Fruit and shoot borer (leucinodes orbonalis), com root worm (Diabrotica spp ), Western com rootworm (diabrotica virgifera), Whitefly (bemisia tabaci), Flouse Fly (Musca Domestica), Green Bottle Fly (Lucilia cuprina), Silk Moth (Bombyx mori), Red Scale (Aonidiella aurantia), Dog heartworm (Dirofilaria immitis). Southern pine beetle (Dendroctonus frontalis), Avocado thrip (Thysanoptera Spp.), Botfly selected from Oestridae spp. and Dermatobia hominis), Florse Fly (Tabanus sulcifrons), Flom Fly (Flaematobia irritans), Screwworm Fly selected from Cochliomyia macellaria (C. macellaria), C. hominivorax, C. aldrichi, or C. minima, Tsetse Fly (Glossina spp.). Warble Fly selected from Flypoderma bovis or Hypoderma lineatum. Spotted lantemfly (Lycorma delicatula), Khapra beetle (Trogoderma granarium), Honeybee mite (Varroa destructor), Termites (Coptotermes formosanus), Hemlock woolly adelgid (Adelges tsugae), Walnut twig beetle (Pityophthorus juglandis), European wood wasp (Sirex noctilio), Pink-spotted bollworm (pectinophora scutigera), Two spotted spider mite (Tertanychus urticae), Diamondback moth (plutella xylostella). Taro caterpillar (spodoptera litura), Red flour beetle (tribolium castaneum). Green peach aphid (Myzus persicae), CottonAttorney Docket No.: 15670-0446WO1Aphid (aphis gossypii), Brown planthopper (nilaparvata lugens), Beet armyworm (spodotera exigua), Western flower thrips (frankliniella occidentals), Codling moth (cydia pomonella), Cowpea weevil (callosobruchus maculatus). Pea aphid (acyrthosiphon pisum). Tomato leafminer (tuta absoluta), Onion thrips (thrips tabaci), or Cotton bollworm (Helicoverpa armigera).

18. The method of claim 16, wherein the insect is a Ceratitis capitata (Mediterranean fruit fly).

19. The method of claim 16, wherein the insect is a mosquito selected from the group consisting of Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus (Aedes triseriatus), Anopheles stephensi, Anopheles albimanus, Anopheles gambiae, Anopheles quadrimaculatus, Anopheles freebomi, Culex species, and Culiseta melanura.

20. A method of identifying the sex of an insect based on a differential sex-specific phenotype, the method comprising:(a) delivering a cisgenic nucleic acid molecule into an insect genome, wherein the nucleic acid molecule comprises a sex-specific splicing module, and wherein the nucleic acid molecule is introduced into an endogenous phenotypic gene of the insect;(b) generating a sex-distinguishable insect population, wherein an insect of the sex-distinguishable insect population comprises the cisgenic nucleic acid molecule; and(c) sorting the insect from the sex-distinguishable insect population based on the differential sex-specific expression of the endogenous phenotypic gene,wherein the endogenous phenotypic gene is functionally expressed in only one sex.

21. The method of claim 20, wherein the sex-specific splicing module activates the expression of the endogenous phenotypic gene.

22. The method of claim 20, wherein the sex-specific splicing module disrupts the expression of the endogenous phenotypic gene.

23. The method of any one of claims 20-22, wherein the sex-specific splicing module comprises an endogenous sex-specific intronic sequence.Attorney Docket No.: 15670-0446WO124. The method of claim 23, wherein the endogenous sex-specific intronic sequence comprises an endogenous transformer (tra) intron.

25. The method of any one of claims 20-24, wherein the endogenous phenotypic gene comprises a white pupae (wp) gene.

26. The method of any one of claims 20-25, wherein the cisgenic nucleic acid molecule is introduced into the endogenous phenotypic gene of the insect by using a gene-editing agent, transposase-mediated insertion, HDR replacement, or recombinase-mediated cassette exchange.

27. The method of claim 26, wherein the gene-editing agent is selected from the group consisting of a CRISPR / Cas9 system, a CRISPR / Casl2 system, a Cast 3 system, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), homology-directed repair, non-homologous end-joining (NHEJ) repair, and recombinases.

28. The method of claim 27, wherein the gene-editing agent comprises CRISPR / Cas9 components.

29. The method of any one of claims 20-28. wherein the sex-distinguishable insect population is generated by cisgenic genetic modification.

30. The method of any one of claims 20-29, wherein the cisgenic nucleic acid molecule is delivered into the insect at the embryo stage of development of the insect.

31. The method of any one of claims 20-30, wherein the insect is sorted as male based on the expression of the endogenous phenotypic gene.

32. The method of any one of claims 20-30, wherein the insect is sorted as female based on the expression of the endogenous phenotypic gene.

33. The method of any one of claims 20-32, wherein the differential sex-specific expression of the endogenous phenotypic gene comprises molecular detection, behavioral phenotype, viability differences, or size or timing differences.Attorney Docket No.: 15670-0446WO134. The method of any one of claims 20-33, wherein the insect is sorted by automated machine-vision sorting, fluorescence-based sorting, or mechanical sex-separation enhanced by phenotype.

35. The method of any one of claims 20-34, wherein the insect is sorted at the larval stage, the pupal developmental stage, or the adult stage.

36. The method of any one of claims 20-35, wherein the insect is a mosquito or a fruit fly.

37. The method of claim 36, wherein the insect is a tephritid fruit fly selected from Medfly (Ceratitis capitata), Mexfly (Anastrepha ludens). Oriental fruit fly (Bactrocera dorsalis), Olive fruit fly (Bactrocera oleae), Melon fly (Bactrocera cucurbitae), Natal fruit fly (Ceratitis rosa), Cherry fruit fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyroni), Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), Oriental Fruit Fly (Bactrocera dorsalis), West Indian fruit fly (Anastrepha obliqua), the New World screwworm (Cochliomyiahominivorax), the Old World screwworm (Chrysomya bezziana), Australian sheep blowfly / greenbottle fly (Lucilia cuprina), the pink bollworm (Pectinophora gossypiella), the European Gypsy moth (Lymantria dispar), the Navel Orange Worm (Amyelois transitella). the Peach Twig Borer (Anarsia lineatelia), the rice stem borer (Tryporyza incertulas), the noctuid moths, Heliothinae, the Japanese beetle (Papilla japonica), White-fringed beetle (Graphognatus spp.), Boll weevil (Anthonomous grandis), the Colorado potato beetle (Leptinotarsa decern lineata), the vine mealybug (Pianococcus ficus), Asian citrus psyllid (diaphorina citri), Spotted wing drosophila (drosophila suzukii), Bluegreen sharpshooter (graphocephala atropunctata), Glassy winged sharpshooter (Flomalodisca vitripennis), Light brown apple moth (Epiphyas postvittana), Bagrada bug (Bagrada hilaris), Brown marmorated stink bug (Halyomorpha halys), Asian Gy psy Moth selected from the group of Lymantria dispar asiatica, Lymantria dispar japonica, Lymantria albescens, Lymantria umbrosa, and Lymantria postalba, Asian longhomed beetle (Anoplophora glabripennis). Coconut Rhinoceros Beetle (Oryctes rhinoceros), Emerald Ash Borer (Agrilus planipennis), European Grapevine Moth (lobesia botrana), European Gypsy Moth (Lymantria dispar), False Codling Moth (Thaumatotibia leucotreta), fire ants selected from Solenopsis invicta Buren, and S. richteri Forel, Old World Bollworm (Flelicoverpa armigera). Spotted Lantemfly (Lycorma delicatula), Africanized honeybee (apis mellifera scutellata), Fruit andAttorney Docket No.: 15670-0446WO1shoot borer (leucinodes orbonalis), com root worm (Diabrotica spp ), Western com rootworm (diabrotica virgifera), Whitefly (bemisia tabaci), Flouse Fly (Musca Domestica), Green Bottle Fly (Lucilia cuprina), Silk Moth (Bombyx mori), Red Scale (Aonidiella aurantia), Dog heartworm (Dirofilaria immitis), Southern pine beetle (Dendroctonus frontalis), Avocado thrip (Thysanoptera Spp.), Botfly selected from Oestridae spp. and Dermatobia hominis), Florse Fly (Tabanus sulcifrons), Flom Fly (Flaematobia irritans), Screwworm Fly selected from Cochliomyia macellaria (C. macellaria), C. hominivorax, C. aldrichi, or C. minima, Tsetse Fly (Glossina spp.), Warble Fly selected from Flypoderma bovis or Hypoderma lineatum, Spotted lantemfly (Lycorma delicatula), Khapra beetle (Trogoderma granarium), Honeybee mite (Varroa destructor), Termites (Coptotermes formosanus), Hemlock woolly adelgid (Adelges tsugae), Walnut twig beetle (Pityophthorus juglandis), European wood wasp (Sirex noctilio). Pink-spotted bollworm (pectinophora scutigera), Two spotted spider mite (Tertanychus urticae), Diamondback moth (plutellaxylostella), Taro caterpillar (spodoptera litura), Red flour beetle (tribolium castaneum), Green peach aphid (Myzus persicae), Cotton Aphid (aphis gossypii), Brown planthopper (nilaparvata lugens), Beet armyworm (spodotera exigua), Western flower thrips (frankliniella occidentalis), Codling moth (cydia pomonella). Cowpea weevil (callosobruchus maculatus), Pea aphid (acyrthosiphon pisum), Tomato leafminer (tuta absoluta), Onion thrips (thrips tabaci), or Cotton bollworm (Helicoverpa armigera).

38. The method of claim 36, wherein the insect is a Ceratitis capitata (Mediterranean fruit fly).

39. The method of claim 36, wherein the insect is a mosquito selected from the group consisting of Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus (Aedes triseriatus), Anopheles stephensi, Anopheles albimanus, Anopheles gambiae, Anopheles quadrimaculatus, Anopheles freebomi, Culex species, and Culiseta melanura.

40. A cisgenic genetically modified insect comprising a nucleic acid molecule comprising a sex-specific splicing module within an endogenous phenotypic gene of the insect.

41. The cisgenic insect of claim 40, wherein the sex-specific splicing module activates the expression of the phenotypic gene.Attorney Docket No.: 15670-0446WO142. The cisgenic insect of claim 40, wherein the sex-specific splicing module disrupts the expression of the phenotypic gene.

43. The cisgenic insect of any one of claims 40-42, wherein the sex-specific splicing module comprises an endogenous sex-specific intronic sequence.

44. The cisgenic insect of claim 43, wherein the endogenous sex-specific intronic sequence comprises an endogenous transformer (tra) intron.

45. The cisgenic insect of any one of claims 40-44, wherein the phenoty pic gene comprises a white pupae (wp) gene.

46. The cisgenic insect of any one of claims 40-45, wherein the insect is sorted as male based on the expression of the phenotypic gene.

47. The cisgenic insect of any one of claims 40-45, wherein the insect is sorted as female based on the expression of the phenotypic gene.

48. The cisgenic insect of any one of claims 40-47, wherein the insect is a mosquito or a fruit fly.

49. The cisgenic insect of claim 48, wherein the insect is a tephritid fruit fly selected from Medfly (Ceratitis capitata), Mexfly (Anastrepha ludens), Oriental fruit fly (Bactrocera dorsalis). Olive fruit fly (Bactrocera oleae), Melon fly (Bactrocera cucurbitae), Natal fruit fly (Ceratitis rosa), Cherry fruit fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyroni). Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), Oriental Fruit Fly (Bactrocera dorsalis), West Indian fruit fly (Anastrepha obliqua), the New World screwworm (Cochliomyia hominivorax), the Old World screwworm (Chrysomya bezziana), Australian sheep blowfly / greenbottle fly (Lucilia cuprina), the pink bollworm (Pectinophora gossypiella), the European Gypsy moth (Lymantria dispar), the Navel Orange Worm (Amyelois transitella), the Peach Twig Borer (Anarsia lineatella), the rice stem borer (Tryporyza incertulas), the noctuid moths, Heliothinae, the Japanese beetle (Papilla japonica), White-fringed beetle (Graphognatus spp.), Boll weevil (Anthonomous grandis), the Colorado potato beetle (Leptinotarsa decern lineata), the vine mealybug (Pianococcus ficus), AsianAttorney Docket No.: 15670-0446WO1citrus psyllid (diaphorina citri), Spotted wing drosophila (drosophila suzukii), Bluegreen sharpshooter (graphocephala atropunctata), Glassy winged sharpshooter (Flomalodisca vitripennis), Light brown apple moth (Epiphyas postvittana), Bagrada bug (Bagrada hilaris). Brown marmorated stink bug (Halyomorpha halys), Asian Gypsy Moth selected from the group of Lymantria dispar asiatica, Lymantria dispar japonica, Lymantria albescens, Lymantria umbrosa, and Lymantria postalba, Asian longhomed beetle (Anoplophora glabripennis), Coconut Rhinoceros Beetle (Oryctes rhinoceros), Emerald Ash Borer (Agrilus planipennis), European Grapevine Moth (lobesia botrana), European Gypsy Moth (Lymantria dispar), False Codling Moth (Thaumatotibia leucotreta), fire ants selected from Solenopsis invicta Buren, and S. richteri Forel, Old World Bollworm (Flelicoverpa armigera), Spotted Lantemfly (Lycorma delicatula), Africanized honeybee (apis mellifera scutellata), Fruit and shoot borer (leucinodes orbonalis), com root worm (Diabrotica spp.), Western com rootworm (diabrotica virgifera), Whitefly (bemisia tabaci), Flouse Fly (Musca Domestica), Green Bottle Fly (Lucilia cuprina), Silk Moth (Bombyx mori), Red Scale (Aonidiella aurantia), Dog heartworm (Dirofilaria immitis). Southern pine beetle (Dendroctonus frontalis), Avocado thrip (Thysanoptera Spp.), Botfly selected from Oestridae spp. and Dermatobia hominis), Florse Fly (Tabanus sulcifrons), Flom Fly (Flaematobia irritans), Screwworm Fly selected from Cochliomyia macellaria (C. macellaria), C. hominivorax, C. aldrichi, or C. minima, Tsetse Fly (Glossina spp.), Warble Fly selected from Flypoderma bovis or Hypoderma lineatum. Spotted lantemfly (Lycorma delicatula), Khapra beetle (Trogoderma granarium), Honeybee mite (Varroa destructor), Termites (Coptotermes formosanus), Hemlock woolly adelgid (Adelges tsugae), Walnut twig beetle (Pityophthorus juglandis), European wood wasp (Sirex noctilio), Pink-spotted bollworm (pectinophora scutigera), Two spotted spider mite (Tertanychus urticae), Diamondback moth (plutella xylostella), Taro caterpillar (spodoptera litura). Red flour beetle (tribolium castaneum), Green peach aphid (Myzus persicae). Cotton Aphid (aphis gossypii). Brown planthopper (nilaparvata lugens), Beet armyworm (spodotera exigua), Western flower thrips (frankliniella occidentalis), Codling moth (cydia pomonella), Cowpea weevil (callosobruchus maculatus). Pea aphid (acyrthosiphon pisum), Tomato leafminer (tuta absoluta), Onion thrips (thrips tabaci), or Cotton bollworm (Helicoverpa armigera).

50. The method of claim 48, wherein the insect is a Ceratitis capitata (Mediterranean fruit fly).Attorney Docket No.: 15670-0446WO151. The method of claim 48, wherein the insect is a mosquito selected from the group consisting of Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus (Aedes triseriatus), Anopheles stephensi, Anopheles albimanus. Anopheles gambiae. Anopheles quadrimaculatus, Anopheles freebomi, Culex species, and Culiseta melanura.

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