阅读说明：本技术 昆虫中的核酸内切酶定性和绝育 (Characterization and sterilization of insect endonucleases ) 是由 N·P·康杜尔 O·S·阿巴里 于 2018-11-19 设计创作，主要内容包括：公开的精确引导的昆虫不育技术(pgSIT)的方法包括在基因修饰的昆虫中指导雄性定性的方法和产生基因修饰的不育雄性昆虫卵的后代的方法。这些方法包括将至少一个核酸序列整合到第一昆虫的基因组中,所述至少一个核酸序列具有至少一个第一引导多核苷酸,所述第一引导多核苷酸靶向雌性特异性生存力所必需的雌性必要的基因组序列,将核酸内切酶引入第二昆虫内,和将第一昆虫和第二昆虫遗传杂交,从而产生表达核酸内切酶和至少一个核酸序列的后代。对于雄性不育,将第二引导多核苷酸靶向雄性特异性不育所必需的雄性不育基因组序列。(Methods of the disclosed precision-guided insect sterility technology (pgSIT) include methods of directing male characterization in genetically modified insects and methods of producing progeny of genetically modified sterile male insect eggs. These methods include integrating at least one nucleic acid sequence into the genome of a first insect, the at least one nucleic acid sequence having at least one first guide polynucleotide that targets female-essential genomic sequences essential for female-specific viability, introducing an endonuclease into a second insect, and genetically crossing the first insect and the second insect, thereby producing progeny that express the endonuclease and the at least one nucleic acid sequence. For male sterility, the second guide polynucleotide is targeted to a male sterility genomic sequence necessary for male-specific sterility.)

1. A method of directing male sex determination in progeny of a genetically modified insect, the method comprising:

integrating at least one nucleic acid sequence into the genome of a first insect, the at least one nucleic acid sequence comprising at least one first guide polynucleotide targeting a female-essential genomic sequence essential for female-specific viability;

introducing an endonuclease into a second insect, said second insect being capable of genetically hybridizing to said first insect; and

genetically crossing the first insect and the second insect, thereby producing progeny comprising the endonuclease and the at least one nucleic acid sequence, male insect eggs from the progeny maturing to adulthood.

2. A method of producing a genetically modified progeny of a sterile male insect egg, comprising the method of claim 1, wherein the at least one nucleic acid sequence further comprises at least one second guide polynucleotide targeting a male sterile genomic sequence necessary for male-specific fertility, wherein genetically crossing the first insect and the second insect produces progeny of a sterile male insect egg.

3. The method of claim 1 or 2, wherein said integrating at least one nucleic acid sequence into the genome of said first insect comprises homozygously integrating into all chromosomal copies in said genome.

4. The method of claim 1 or 2, wherein said integrating said at least one nucleic acid sequence comprises introducing said at least one nucleic acid sequence into said first insect during an embryonic stage.

5. The method of claim 1 or 2, wherein the at least one first guide polynucleotide and the at least one second guide polynucleotide each comprise at least one guide ribonucleic acid (gRNA).

6. The method according to claim 1 or 2, wherein the female essential genomic sequence comprises a gene or female specific exons essential for female specific viability or female specific development and/or female specific viability.

7. The method of claim 1 or 2, wherein the at least one first guide polynucleotide comprises more than one first guide polynucleotide, each of the first guide polynucleotides targeting different regions of the same female-essential genomic sequence that is essential for female-specific viability.

8. The method of claim 1 or 2, wherein the at least one first guide polynucleotide comprises more than one first guide polynucleotide, each of the first guide polynucleotides targeting different female-essential genomic sequences essential for female-specific viability.

9. The method of claim 1 or 2, wherein the female-essential genomic sequence is a gene or splice variant of a gene selected from the group consisting of sex-determining lethal gene (Sxl), transgene (Tra), diplotene (Dsx), homolog thereof, ortholog thereof, paralog thereof, and combination thereof.

10. The method of claim 1 or 2, wherein the at least one first guide polynucleotide comprises more than one first guide polynucleotide, each of the first guide polynucleotides targeting a different gene selected from the group consisting of Sxl, Tra, Dsx, homologs thereof, orthologs thereof, and paralogs thereof.

11. The method of claim 10, wherein the more than one first guide polynucleotide comprises two first guide polynucleotides, each of the first guide polynucleotides targeting a different gene selected from the group consisting of Sxl, Tra, Dsx, homologs thereof, orthologs thereof, and paralogs thereof.

12. The method of claim 10, wherein the more than one first guide polynucleotide comprises two first guide polynucleotides, each of the first guide polynucleotides targeting a different gene selected from the group consisting of Sxl, Dsx, homologs thereof, orthologs thereof, and paralogs thereof.

13. The method of claim 2, wherein the male sterile genomic sequence is a gene selected from the group consisting of β Tubulin85D (β Tub), fuzzy onions (Fzo), protamine a (prota), and spermatocyte block (Sa).

14. The method of claim 1 or 2, wherein: introducing the endonuclease into the second insect comprises homozygously integrating a gene encoding the endonuclease when the second insect is male, and introducing the endonuclease into the second insect comprises homozygously or heterozygously integrating a gene encoding the endonuclease or depositing an endonuclease protein into the second insect when the second insect is female.

15. The method of claim 1 or 2, wherein said introducing an endonuclease into a second insect comprises introducing the endonuclease into the second insect during an embryonic stage.

16. The method of claim 1 or 2, wherein the endonuclease comprises a CRISPR-associated sequence 9(Cas9) endonuclease or a variant thereof, a CRISPR-associated sequence 13(Cas13) endonuclease or a variant thereof, a CRISPR-associated sequence 6(Cas6) endonuclease or a variant thereof, a CRISPR (Cpf1) endonuclease or a variant thereof from prevotella and francisella 1 or a CRISPR (Cms1) endonuclease or a variant thereof from microgenerates and smithlla 1.

17. The method of claim 1 or 2, wherein the endonuclease comprises Streptococcus pyogenes Cas9(SpCas9), Staphylococcus aureus Cas9(SaCas9), Francisella newcastle Cas9(FnCas9), or a variant thereof.

18. The method of claim 17, wherein the variants thereof comprise a pro-spacer sequence adjacent motif (PAM) SpCas9(xCas9), high fidelity SpCas9(SpCas9-HF1), high fidelity SaCas9, or high fidelity FnCas 9.

19. The method of claim 1 or 2, wherein the endonuclease comprises a Cas fusion nuclease comprising a Cas9 protein or variant thereof fused to a fokl nuclease or variant thereof.

20. The method of claim 19, wherein the variant thereof comprises a catalytically inactive Cas9 (dead Cas 9).

21. The method according to claim 1 or 2, wherein the endonuclease is Cas9, Cas13, Cas6, Cpf1, CMS1 protein or any variant thereof derived from methanococcus marinus C7, corynebacterium diphtheriae, corynebacterium hyperboloides YS-314, corynebacterium glutamicum (ATCC 13032), corynebacterium glutamicum R, corynebacterium chrysotile (DSM 44385), mycobacterium abscessus (ATCC19977), nocardia suberoyheidoides IFM10152, rhodococcus erythropolis PR4, rhodococcus giae RHA1, rhodococcus opaque B4(uid36573), thermobacter cellulolyticus 11B, arthrobacter chlorophenol A6, Kribbellaflavida (DSM 17836, uid43465), thermomonospora curvata (DSM43183), bifidobacterium bifidum Bd1, bifidobacterium longum 3810, klebsiella reducta (DSM 20476), pseudomonas persolanacearum 4676, pseudomonas fragilis 4634, pseudomonas solanacearum 4634, DSM 4634, C t B943B 9, C1, C Flavobacterium psychrophilum JIP 0286, Exmenoptera muciniphila (ATCC BAA 835), photosynthetic roseobacter (DSM 13941), Curvularia RS1, Synechocystis PCC6803, Microtracella Pei191, uncultured termite type 1 bacterial species Rs D17, filobacillus succinogenes S85, Bacillus cereus (ATCC 10987), Listeria innocua, Lactobacillus casei, Lactobacillus rhamnosus GG, Lactobacillus salivarius UCC118, Streptococcus agalactiae-5-A909, Streptococcus agalactiae NEM316, Streptococcus agalactiae 2603, Streptococcus Martensis GGS 124, Streptococcus equi subsp MGCS10565, Streptococcus gallic acid UCN34(uid46061), Streptococcus gordoniae subset CH1, Streptococcus mutans 2025(uid NN 46353), Streptococcus mutans, Streptococcus pyogenes 1 GAS, Streptococcus pyogenes MGAS5005, MGMGAS 1026, Streptococcus pyogenes AS 29, Streptococcus pyogenes AS 61870, Streptococcus pyogenes AS 2090, Streptococcus pyogenes AS 70, Streptococcus mutans AS 46353, Streptococcus pyogenes MGAS315, Streptococcus pyogenes SSI-1, Streptococcus pyogenes MGAS10750, Streptococcus pyogenes NZ131, Streptococcus thermophilus CNRZ1066, Streptococcus thermophilus LMD-9, Streptococcus thermophilus LMG 18311, Clostridium botulinum A3 Loch Maree, Clostridium botulinum B Eklund17B, Clostridium botulinum Ba 4657, Clostridium botulinum Flangeland, Clostridium cellulolyticum H10, Fengolds Daphnevus (ATCC 29328), Eubacterium procumbens (ATCC33656), Mycoplasma gallisepticum, Mycoplasma mobilis 163K, Mycoplasma penonii, Mycoplasma synoviae 53, Streptococcus moniliformis (DSM12112), Rhizobium bradyrhizogenes BTai1, Nitrosum Han X14, Pseudomonas rhodopseudomonas Bib 18, Pseudomonas Bib 5, Gluconotis cleaner DS-1, Corynebacterium rhinoceros DFL 12, Lactobacillus diazotrophicus FAPlJ 5, and Acetobacter diazonianum, Azospirillum azotobacter B510(uid46085), Rhodospirillum rubrum (ATCC 11170), parachloroaniline TPSY (uid29975), Epimedium stercoralis EF01-2, Neisseria meningitidis 053442, Neisseria meningitidis alpha 14, Neisseria meningitidis Z2491, Desulfovibrio halobacter halophilus DSM 2638, Campylobacter jejuni Delley subspecies 26997, Campylobacter jejuni 81116, Campylobacter jejuni, Campylobacter erythraea RM2100, Spirobacter hepaticum, Wollelium succinogenes, Tolypocladium olgensis DSM 9187, Pseudoalteromonas maxima T6c, Shewanella pelalaana (ATCC 700345), Legionella pneumophila, Actinobacillus succinogenes 130Z, Pasteurella multocida, Francisella tularensis noviviida U112, Francisella tularensis tularensis, Francisella lathyris, Francisella calophylla 341198, Francisella calospirillum Y468 or W598.

22. The method of claim 1 or 2, wherein the first insect and the second insect are the same insect or two different insects capable of mating and are in a order selected from the order diptera, lepidoptera, or coleoptera.

23. The method of claim 22, wherein the insect is a mosquito from the genus anopheles, aedes, anopheles, or culex.

24. The method of claim 23, wherein the mosquito is selected from the group consisting of aedes aegypti, aedes albopictus, aedes tripartite haranthidae (aedes trifoliata), anopheles stephensi, anopheles leucatensis, anopheles gambiae, anopheles quadrimaculata, anopheles fantasus, culex pipiens, and aedes nigricans.

25. The method of claim 22, wherein the insect is selected from the group consisting of: drosophila melanogaster (Medfly) (Medflash (Ceratota)), Mexfly (Mexico Fruit Fly (Anastrepluhalus)), Oriental Fruit Fly (Oriental Fruit Fly) (Bactrocera dorsalis), Olive Fruit Fly (Olive Fruit Fly) (Bactrocera oleae), Melon Fruit Fly (Melon Fly) (Bactrocera cucurbitae), Natales Fruit Fly (Natal Fruit Fly) (Natatata Fruit Fly (Ceratorsia), Cherry Fruit Fly (Cherry Fruit Fly) (Rhagogues Fruit Fly)), Queenstem Fruit Fly (Queenstem Fruit Fly) (Bactrocera cerealis (Bactrocera), Fruit Fly (Cherry Fruit Fly) (Hazary (Hazara), and Hazara Fruit Fly (Hazara Fruit Fly) (Hazara Fruit Fly (Hazara ratio)), Queen Fruit Fly (Queenfly) (Bactrocera Fruit Fly) (Hazara), Fruit Fly (Fruit Fly) (Hazara Fruit Fly (Hazara), Hazara Fruit Fly (Hazara Fruit Fly) (Hazara Fruit Fly (Hazara) and Hazara Fruit Fly (Hazara) in Hazara), Hazara Fruit Fly (Hazara Fruit Fly) (Hazara Fruit Fly (Hazara) and Hazara Fruit Fly) (Hazara Fruit Fly) (Hazara) and Hazara Fruit Fly (Hazara Fruit Fly) (Hazara) of Hazara, Hazara, New World Worm (New World Worm screw word) (trypanosoma japonicum (cochlioma hominivorax)), old World Worm (old World Worm screw word) (chrysomyia melanogaster (Chrysomya benthia), Australian sheep green-head/green fly (austrian sheep yellow/green fly) (Lucilia cuprina), pink bollworm (ping bollworm) (pink bollworm (pecnophora gossypiella)), european gypsy (european gypsy moth) (Lymantria), Orange Worm (the naval Orange Worm) (Orange Borer (amyosis), pink bollworm (pink bollworm) (Peach Borer (pink bollworm)), Japanese cabbage moth (pink moth) and pink moth (pink moth) (ostrinia nubilalis), pink moth (pink moth) (ostrinia nubila), black rice stem Borer (pink moth) (ostrinia), black rice stem Borer (pink moth) (ostrinia), black rice stem Borer (pink stem Borer) (ostrinia), black rice stem Borer (pink) (ostrinia), black rice stem Borer (pink) (ostrinia), black Borer (pink) (pink) moth (pink stem Borer), black rice stem Borer) (pink) (pink) moth) stem Borer), black rice stem Borer) and pink (pink) stem Borer), black rice stem Borer), Cotton Boll weevil (Boll weevil) (cotton Boll weevil (Anthonomous grandis)), potato beetle (the Colorado patato beetle) (Leptinota decemlineata (Leptinota)), grape mealybug (the vine mealybug) (Ficus carica (Planococcus fiscus), Asian citrus psylla (Asian citrus psylla) (diaphorina citri), Spotted wing drosophila (Spotted drosophila) (cherry fruit fly (drosophila sukii)), blue green cicada (blue green sharpsophilter) (apple cicada (Graphophalolaatrix), glass leaf leafhopper (Glywig) (glass leafbud), Brown apple moth (apple moth), apple moth (apple leaf moth) (apple moth), apple leaf moth (apple leaf moth), apple leaf moth (Brown apple leaf moth) (apple leaf moth)), Brown apple leaf moth (apple leaf moth) (apple leaf moth (Brown apple leaf moth) (apple leaf moth) (Brown apple leaf moth) (apple leaf moth (Brown apple leaf moth)), green moth (Brown apple leaf moth) (Brown apple leaf moth) (sweet)), green leaf moth (Brown apple leaf moth) (sweet)) Lymantria umbrosa and Lymantria posttala), Asian beetles (Aspian longihorned beetles) (Anoplophora glabripennis), Coconut shell beetles (Coconut Rhinoceros beetles) (Coconut shell beetles) (Oryctes rhinus), white wax tree Borer (emery apple narrow Giardia (Agrilspelinis)), European grape Moth (European Gypsy Moth) (grape leaf Moth (lobemilia borna)), European Gypsy Moth (dance Moth), fake apple leaf Moth (False apple leaf Moth) (apple heterostemma leaf Moth (Thaumatobotrya)), fire ants (selected from the group consisting of red fire Moth (Solenopsis virginica) and white bee (Fabricius) and African yellow cabbage Moth (Wallicheria meldoni)), yellow fly larva (yellow cabbage Moth (Wallichen) and yellow cabbage Moth (yellow cabbage Moth) (African yellow cabbage Moth (yellow cabbage Moth) Corn rootworm (corn rootworm) (Diabrotica Spp.), Western corn rootworm (Western corn rootworm) (corn rootworm (Diabrotica virgifera)), Whitefly (bemisia tabaci), HouseFly (HouseFly) (Musca domestica), Green Fly (Green bot Fly) (Lucilia cuprina), Silk Moth (Silk Moth) (Bombyx mori), Red clam (Red Scale) (aonidia aurantia), Dog heart Silk Fly (Dog heart worm) (diaphania), southern pine small Fly (southern pine Moth) (southern yellow), and Western pink (Western blot), some species of blowfly (blowfly), some species of blowfly (blowfly), silkworm Moth (silkworm Moth mole, silkworm (silkworm, silkworm), silkworm (Moth), some species of blowfly (blowfly), south pine Moth, blowfly (blowfly), some species of blowfly (blowfly, blow, Trypanosoma manophilum (c.hominivorax), c.aldrich or c.minima), Tsetse Fly (Tsetse Fly) (glomus sp.), dermatosis Fly (Warble Fly) selected from dermomyia bovis (Hypoderma bovis) or dermomyia striata (Hypoderma linedefect), Ceratopsis punctatus (spoted landmass) (Lycocarpus punctatus)), Ceratopsis punctatus (Khapaberte) (Tropidus gratus), Melissa mellifera (Honeybee), Melastoma punctatus (Tetranychus urticae), Termite (Coptoterus punctatus), Pyrenopsis punctatus (Pilotosus)), Meloidea punctatus (Pimpinella punctatus), Periploca punctatus (Pilotoides) (Pimpinus punctatus (Pimenta punctatus)), Meloidea punctatus (Pilus punctatus), Pilotoides (Pimpinus punctatus (Pilus punctatus) (Pimenta punctatus) (Pilus punctatus)), Melongopus punctatus (Pilus punctatus) (Pilus punctatus)), Coptophycus (Pilus punctatus)), Coptophycus (Pilus punctatus) (Pilus punctum), Pilus punctatus (Pilus punctatus) (Pilus punctum), Pilus punctum) of Pilus punctum punctatus (Pilus punctum), Pilus punctatus (Pilus punctum, Taro caterpillar (tarocaterpilar) (prodenia litura), Red flower beetle (Red flower beetle) (trichoderma castaneum), Green peach Aphid (Green peach Aphid) (Myzus persicae), Cotton Aphid (Cotton Aphid), Brown planthopper (Brown planthopper) (nilaparvata lugens), Beet armyworm (Beet armyworm) (spodiotera exigua (spodoptera exigua)), western flower Thrips (Western flower Thrips) (frankliniella occidentalis), Codling moth (cydia pomonella), Cowpea elephant (Cowpea elephant), Pea aphid (Pea aphid), Tomato leaf miner (Tomato leaf miner)), Onion Thrips (Thrips tabaci)) and Cotton bollworm (Cotton bollworm) (Cotton bollworm (Helicoverpa armigera)).

26. Progeny of an insect egg comprising up to 100% of a male insect egg produced by the method of any one of claims 1-25.

27. Progeny of an insect egg comprising up to 100% sterile male insect eggs produced by the method of any one of claims 2-25.

28. A genetically modified sterile male insect produced by the method of any one of claims 2-25, which is capable of increasing the ratio of unhatched eggs by mating with a wild-type female insect.

29. The genetically modified sterile male insect of claim 28, wherein the genetically modified sterile male insect has a lifespan equal to or longer than its corresponding wild-type male insect.

30. A method of reducing a wild-type insect population comprising introducing genetically modified sterile males into a wild-type insect population, the genetically modified sterile males produced by the method of any one of claims 2-25.

Background

The mass production and release of sterile males, known as insect sterility technology (SIT), has historically been used to control and eradicate pest populations dating back to the mid-thirties of the twentieth century. Previous methods relied on DNA damaging agents for sterility, substantially reducing the overall health (fitness) and mating competitiveness of released males. To overcome these problems, microorganism-mediated sterility techniques such as the wolbachia-based Insect Incompatibility Technique (IIT) and modern genetic SIT-like systems such as the Release of Insects carrying Dominant Lethal genes (insect Dominant Lethal Release technique, Release of Insects harvesting a Dominant Lethal, RIDL), and other methods of gene killing females to Release fertile males, such as female-specific RIDL (fsridl) and autosomal linked X chromosome shredders (shredders) have been developed. While these first generation genetic SIT technologies represent a significant advance, IIT strict requirements do not release infected females, which are difficult to achieve in the field, and the use of tetracycline ablation (ablate) microbiota is known to compromise the wellness of RIDL/fsRIDL males, and X chromosome mills can in principle only be developed in species with heteroleptic chromosomes, limiting widespread applicability to other species. Therefore, it would be logically advantageous to employ more efficient SIT-based techniques that can be deployed as eggs — only sterile males survive.

Disclosure of Invention

Aspects of embodiments of the present disclosure relate to methods comprising a precision-guided insect sterility technology (pgSIT).

In some embodiments of the disclosure, a method of directing male sex in a genetically modified insect comprises: integrating at least one nucleic acid sequence into the genome of the first insect, the at least one nucleic acid sequence having at least one first guide polynucleotide targeting a female-essential genomic sequence essential for female-specific viability; introducing an endonuclease into a second insect, the second insect being capable of genetically hybridizing to the first insect; and genetically crossing the first insect and the second insect, thereby producing progeny expressing the endonuclease and the at least one nucleic acid sequence, from which progeny a male insect egg matures to adulthood.

In some embodiments of the disclosure, a method of producing progeny of genetically modified sterile male insect eggs comprises: integrating at least one nucleic acid sequence into the genome of the first insect, the at least one nucleic acid sequence having at least one first guide polynucleotide targeting a female-essential genomic sequence essential for female-specific viability; introducing an endonuclease into a second insect capable of genetically hybridizing to the first insect, wherein the at least one nucleic acid sequence further comprises at least one second guide polynucleotide that targets a male-sterile genomic sequence necessary for male-specific sterility; and genetically crossing the first insect and the second insect to produce progeny of the genetically modified sterile male insect egg.

In some embodiments of the disclosure, integrating the at least one nucleic acid sequence into the genome of the first insect comprises homozygously integrating into all chromosomal copies in the genome. In some embodiments, integrating at least one nucleic acid sequence comprises introducing the at least one nucleic acid sequence into the first insect during the embryonic stage.

In some embodiments of the present disclosure, the at least one first guide polynucleotide and the at least one second guide polynucleotide each comprise at least one guide ribonucleic acid (gRNA).

In some embodiments of the disclosure, the female-essential genomic sequence comprises a gene or female-specific exon that is essential for female-specific viability.

In some embodiments of the disclosure, the at least one first guide polynucleotide comprises more than one first guide polynucleotide, each first guide polynucleotide targeting a different region of the same female-essential genomic sequence that is essential for female-specific viability.

In some embodiments of the disclosure, the at least one first guide polynucleotide comprises more than one first guide polynucleotide, each first guide polynucleotide targeting a different female-essential genomic sequence essential for female-specific viability.

In some embodiments of the disclosure, the female-essential genomic sequence is a gene or splice variant of a gene selected from the group consisting of a sex-determining lethal gene (Sxl), a transgene (Tra), a diplotene (Dsx), a homolog thereof, an ortholog thereof, a paralog thereof, or a combination thereof.

In some embodiments of the disclosure, the at least one first guide polynucleotide comprises more than one first guide polynucleotide, each first guide polynucleotide targeting a different gene selected from Sxl, Tra, or Dsx, including a homolog thereof, an ortholog thereof, or a paralog thereof.

In some embodiments of the disclosure, the more than one first guide polynucleotide comprises two first guide polynucleotides, each targeting a different gene selected from Sxl, Tra or Dsx, including a homolog thereof, an ortholog thereof, or a paralog thereof.

In some embodiments of the disclosure, the more than one first guide polynucleotide comprises two first guide polynucleotides, each targeting a different gene selected from Sxl or Dsx, including a homolog thereof, an ortholog thereof, or a paralog thereof.

In some embodiments of the disclosure, the male sterile genomic sequence is a gene selected from β Tubulin85D (β Tub), fuzzy onions (Fzo), protamine a (prota), or spermatocyte block (Sa), including a homolog, ortholog thereof, or paralog thereof.

In some embodiments of the disclosure, introducing the endonuclease into the second insect comprises homozygously integrating the gene encoding the endonuclease when the second insect is male, and introducing the endonuclease into the second insect comprises homozygously or heterozygously integrating the gene encoding the endonuclease or depositing the endonuclease protein into the second insect when the second insect is female.

In some embodiments of the disclosure, introducing the endonuclease into the second insect comprises introducing the endonuclease into the second insect during the embryonic stage.

In some embodiments of the disclosure, progeny of genetically modified insect eggs include up to 100% of male insect eggs produced according to the methods of the disclosure.

In some embodiments of the disclosure, progeny of genetically modified insect eggs include up to 100% sterile male insect eggs produced according to the methods of the disclosure.

In some embodiments, genetically modified sterile male insects produced according to the methods of the present disclosure are capable of increasing the ratio of unhatched eggs by mating with wild-type female insects.

In some embodiments of the disclosure, a method of reducing a wild-type insect population comprises introducing genetically modified sterile males produced according to a method of the disclosure into a wild-type insect population.

Drawings

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee. The drawings illustrate example embodiments of the disclosure and, together with the description, serve to explain the principles of the disclosure.

Figure 1A is a schematic of the use of the two components of the binary CRISPR/Cas9 system, endonuclease Cas9 and guide ribonucleic acid (gRNAs) (blue or green target-specific sequences) pgSIT, maintained as separate homozygous lines whose hybridization results in concurrent or simultaneous gene knockout of the gene required for female viability and the gene required for male sterility, resulting in F only, in accordance with embodiments of the present disclosure₁Survival of sterile males.

Figure 1B is a schematic representation of gender-specific alternative splicing in the sxl, Tra, and dsx genes regulated by female expression of Sxl (green) and Tra (yellow) proteins (grey line), according to embodiments of the present disclosure; disruption of female-specific exons of the key sex-determining genes sxl, tra, and dsx disrupts female development; and pgSIT exon targets are indicated by yellow crosses.

Fig. 1C presents a schematic diagram of all constructs engineered according to an embodiment of the present disclosure, wherein the functional construct and flies are deposited at the adddge. The gene name and gRNA target site sequence are presented in frame. The coding sequence of SpCas9 flanked at both ends by two Nuclear Localization Signals (NLS) and at the C-terminus by a self-cleaving T2A peptide with eGFP coding sequence served as a visual indicator of Cas9 expression.

Fig. 1D is a fluorescence stereomicroscope image of three new homozygous lines expressing streptococcus pyogenes Cas9(SpCas9) engineered according to embodiments of the present disclosure. Three drosophila lines were developed that support expression of SpCas9 in either the strict germ line or in germ lines with somatic cells. Use of-mediated integration, insertion of Nanos-Cas9(nos-Cas9), vasa-Cas9(vas-Cas9) and Ubiquitin-63E (Ubi-Cas9) at the same position of chromosome 3. The Opie2-dsRed transgene served as a transgene marker and self-cleaving T2A-eGFP sequence attached to the 3' -end of SpCas9 coding sequence, providing an indication of Cas9 expression shown in fig. 1C. Expression levels of dsRed and eGFP in each Cas9 line were compared to wild type (wt) flies. Cas9-T2A-eGFP expression is mainly limited to female germ lines in nos-Cas9 and vas-Cas9, with strong expression in nos-Cas 9. Ubi-Cas9 supported the strongest expression of Cas9 measured by eGFP in both female and male germ lines as well as in somatic cells.

FIG. 1E shows F hybridized to an engineered parent insect according to an embodiment of the disclosure₁Bar graph of mean sex frequency in offspring. The top two plots depict the sex frequency of a two-way control cross from a homozygous sgRNA line with wild type (wt), indicating that fertile females and males (male and female) are present in similar ratios, but no sterile hermaphroditism was identified (□). Fertile females are shown in pink, fertile males in blue, sterile females in orange, and sterile males in gray. The lower two panels show the gender frequency from crosses of homozygous nos-Cas9(nos-Cas9) with wild type (control) and four homozygous sgRNA lines (experiment). 100% trans-hybrid sgRNA independent of maternal or paternal Cas9 inheritance^SxlFemale parent is lethal, 100% of trans-heterozygous sgrnas^TrAnd sgRNA^DsxFMale parent is masculinized to sterile hermaphroditic □, and 100% trans hybrid sgRNA^βTuThe majority is sterile. Sex frequency and fertility in trans-heterozygotes were compared to those in the corresponding progeny of nos-Cas9 (solid line) or sgRNAs (dashed line) and control crosses of wild-type flies. Each bar graph shows the mean gender frequency and one standard deviation. Statistical significance was calculated using the t-test, assuming that the variances were not equal, and for male sterility P-values (red) of the list were calculated using the pearson chi-square test. (P)>0.001***)。

FIG. 1F is a schematic diagram of an embodiment according to the present disclosureF from a cross between homozygous single gRNA (sgRNA/sgRNA) and homozygous nos-Cas9(nos-Cas9/nos-Cas9)₁Table of offspring.

Fig. 1G is a table of genotyping loci targeted by grnas, where insertions/deletions were found in trans-heterozygous flies (red text), using methods according to embodiments of the disclosure.

Fig. 2A shows trans-heterozygous F resulting from a cross between double grna (dsrna) and Cas9 homozygous lines according to embodiments of the present disclosure₁Bar graphs of sex (, female), (male) and □ hermaphroditic) frequencies of progeny the three double guide RNAs (dgRNAs) targeting sxl, tra or dsx, respectively, in combination with β Tub, were crossed bidirectionally with three Cas9 lines driven by the nas (nos), vasa (vas) and Ubiquitin-63E (Ubi) promoters and sufficiently to ensure complete penetrance rates of both female lethality/masculinization and male sterility in each cross as indicated in FIGS. 1C-1D>0.01**，P>0.001***)。

Fig. 2B is F from a cross between homozygous double gRNA (sgRNA/dgRNA) and homozygous Cas9(Cas9/Cas9) lines according to embodiments of the present disclosure₁Table of offspring.

FIG. 2C is a data sheet showing the order of targeted genes in the sex determination pathway in offspring (top panel) and corresponding knockout phenotypes (using images) in accordance with embodiments of the present disclosure phenotype of dgRNA directs knockout and hermaphroditic morphology compared to wild-type male and female β Tub, Sxl knockout disappear during pupal stage as indicated in FIGS. 2D-2E^βTub,Tra/+; nos-Cas9/+ hermaphroditic (□), but not dgRNA^βTub,DsxF/+; nos-Cas9/+ □, with sexual combs-see enlarged internal inset。

FIG. 2D shows a bar graph showing estimated nos-Cas9/nos-Cas9 female and dgRNA homozygous by cross, according to an embodiment of the present disclosure^βTub,Sxl/dgRNA^βTub,SxlVertically produced dgRNA^βTub,Sxl/+; the hatchability (percentage) of nos-Cas9/+ eggs was not statistically different from that of wild type (wt) eggs indicated in table 2 (example 6). Statistical significance was calculated using the t-test, exhibiting unequal variance. (P)<0.05^NS，P>0.001***)。

FIG. 2E shows a bar graph showing incubated dgRNAs according to an embodiment of the present disclosure^βTub,Sxl/+; ratio of different results for nos-Cas9/+ larvae, 50 hatched larvae batches were fed to adults, whose gender or time to death development was recorded as shown in table 3 (example 6). Most additional larval mortality occurs during pupal transformation, and the percentage of pupal death is not statistically different from the wild-type female percentage. Statistical significance was calculated using the t-test, exhibiting unequal variance. (P)<0.05^NS，P>0.001***)。

Fig. 2F is a table of phenotypic characteristics of trans-heterozygous flies carrying Cas9 and double grnas (dsrnas), according to embodiments of the disclosure.

Figure 2G shows a microscope image of variable expression of the number of sex comb bristles in β Tub, Tra knockout □ according to an embodiment of the present disclosure.

Fig. 2H shows a microscope image of the internal reproductive organs of a wild-type female (top panel) according to an embodiment of the present disclosure: two ovaries (ov), seminiferous vesicles (sr), double fertilized sacs (sp), two accessory glands (ag) and uterus (ut), and dgRNA^βTub,Tra/+; nos-Cas9/+ □ hermaphrodite flies (lower panel) have an undeveloped ovary, and organs similar to the male accessory gland.

FIG. 2I is an agarose gel image of transcripts amplified according to an embodiment of the disclosure, showing that both male and female splice variants of the Dsx gene are expressed in β Tub, Tra gene knockout hermaphrodism RT-PCR for evaluation versus wild type (wt) femalesMale (female), wild type male (male) and dgRNA^βTub,TraBoth female and male specific dsx transcripts were identified in β Tub, Tra □. Molecular Ladders (ML) and No Template Controls (NTC) indicating double stranded DNA.

FIG. 2J shows a dgRNA according to an embodiment of the disclosure^βTub,DsxF/+; nos-Cas9/+ □ sex flies developed only a single ovary (ov) not normally connected to the oviduct as indicated and microscopic images of organs similar to the male-specific accessory gland (ag).

Fig. 2K shows a dgRNA with indicated testis (ts) and adrenal (ag) according to embodiments of the present disclosure^βTub,Sxl/+; microscopic image of the male internal reproductive system in nos-Cas9/+ "drosophila.

FIG. 2L shows a dgRNA with testis (ts) and adrenal (ag) indicated, according to embodiments of the present disclosure^βTub,Sxl/+; microscopic image of the male internal reproductive system in nos-Cas9/+ "drosophila.

FIG. 2M shows a microscope image of wild type wt testis (left panel) with elongated cysts with the dgRNA in them, according to an embodiment of the disclosure^βTub,Sxl/+; mature sperm cells not found in nos-Cas9/+ testis (ts) (as indicated here and in FIGS. 2K-2L).

FIG. 2N is a diagram of a trans-heterozygous dgRNA, according to an embodiment of the disclosure^βTub,SxlA schematic of the sequence information of the β Tubulin85D (β Tub) target in nos-Cas9/+ (double knockout) sterile males (long), showing precise chimeric (mosaic) insertion/deletion at the β Tub target site the top panel presents the positions of the gRNA target site and primers used for PCR relative to the genetic structure of the targeted gene reading the sequences from both ends infers the diversity of the template, which is specifically located at the site targeted with gRNA in sterility, while wild type has a single allele without any sequence ambiguity.

FIG. 2O is a diagram relating to trans-shuffling, in accordance with embodiments of the present disclosuredgRNA^βTub,Sxl/+; schematic representation of sequence information for sex-determining lethal gene (Sxl) target in nos-Cas9/+ (double knockout) sterile males (holy), shown on the same dgRNA^βTub,Sxl/+; chimeric insertions/deletions identified at the Sxl target site in nos-Cas9/+ sterile males (male) and may be associated with pupal lethality in trans heterozygous females as observed in fig. 2D-2E. The upper panel presents the gRNA target sites and the location of the primers used for PCR relative to the genetic structure of the targeted gene. The diversity of the template was deduced from the reading of the sequences from both ends, the template was specifically located at the site targeted by gRNA in infertility, whereas the wild type had a single allele without any sequence ambiguity.

FIG. 2P is a diagram of a trans-heterozygous dgRNA, according to an embodiment of the present disclosure^βTub,Tra/+; schematic representation of sequence information for the transgene (Tra) target in nos-Cas9/+ double knockout sterile males (male and female) and male (□), showing localization by the dgRNA^βTub,TraChimeric insertions/deletions at the Tra sites targeted by the double guide rna (dgrna). The top panel shows the positions of the gRNA target and primers used for PCR relative to the genetic structure of the targeted gene. The diversity of the template was deduced from the sequence reads at both ends, the template was specifically located at the sites targeted with grnas in sterility and □, but the wild type had a single allele without any sequence ambiguity at both sites.

FIG. 2Q is a diagram of a dgRNA, according to an embodiment of the present disclosure^βTub,DsxF/+; trans-hybrid dgRNA in nos-Cas9/+ sterility and □^βTub,DsxFSchematic representation of sequence information for the dual sex gene (DsxF) target in the dual gRNA showing that chimeric insertions/deletions were identified at the DsxF site target. The top panel shows the positions of the gRNA target and primers used for PCR relative to the genetic structure of the targeted gene. The diversity of the template was deduced from the sequence reads at both ends, the template was specifically located at the sites targeted with grnas in sterility and □, but the wild type had a single allele without any sequence ambiguity at both sites.

Fig. 3A shows a bar graph representing genetic quantification of dominant effects caused by maternal loading of Cas9, where the genotypes, sex frequencies, and fertility of flies generated by positive and negative crosses between homozygous dgRNA and heterozygous Cas9 flies are represented by pink, blue, orange, or gray solid or striped bars shown in the legend. Progeny that cross with the hybrid male parent Cas9 are shown on the left panel, and progeny that cross with the hybrid female parent Cas9 are shown on the right panel. Each bar graph shows the mean gender frequency and one standard deviation. Statistical significance was calculated using the t-test, exhibiting unequal variance. (P >0.01, P > 0.001). The striped bar indicates Cas9 as inheritance of the gene, while the solid bar indicates inheritance of the + allele.

Fig. 3B is a schematic table showing combinations of genotypes and maternal/zygote contributions in embryos and their exonic rates, according to embodiments of the present disclosure.

FIG. 3C is F from a cross between a homozygous double gRNA (dgRNA/dgRNA) and a heterozygous Cas9(Cas9/TM3, Sb) according to embodiments of the disclosure₁Table of offspring.

Fig. 3D is a schematic table showing that the accumulation of high levels of biallelic chimeras (BMs) throughout insect development leads to loss of gene function at the organism level and ensures complete penetrance of the indicated induced phenotypes: lethality (lethal biallelic chimera (LBM)) (pink box), feminization, or male sterility. Complementation of gene function in some cells by uncut wild type allele (light green box) and resistance allele generated by NHEJ (yellow box) was not sufficient to rescue induced phenotype at the organism level, and thus 100% of trans-heterozygous offspring had induced phenotype. As cells divide, the box becomes smaller and richer.

FIG. 4A is an estimation of dgRNA according to an embodiment of the present disclosure^βTub,Sxl/+; schematic of experimental setup for nos-Cas9/+ sterile males (labeled with red) to compete with wild type males to ensure mating competitiveness for mating with wild type females. The mated females are resistant to the next mating for about 24 hours, and the mating success of the sterile males is assessed by reduced fertility (e.g., by increasing the hatchability of the eggs).

Fig. 4B shows a bar graph showing the percentage of eggs laid and eggs hatched, where the number of eggs laid was normalized to the highest egg number (n-199) to convert them to percentiles as indicated in the table of fig. 4C. The presence of one sterile male leads to a significant reduction in female fertility (#3 vs #2), which can be explained by the removal of one wild-type male (#2 vs # 1). Statistical significance was calculated using the t-test, comparing group #3 with group #2 and group #1, and presented that the variances were not equal (P >0.003 x, P >0.0001 x).

FIG. 4C is a dgRNA as compared to a wild-type male based on laid eggs, unhatched eggs, and hatched eggs, according to an embodiment of the present disclosure^bTub,Sxl/+; table of mating competence of nos-Cas9/+ males with indicated crosses.

Fig. 4D is a graph of wild-type male (blue line) and two types of dgrnas with paternal (red line) or maternal (green line) Cas9 inheritance, according to embodiments of the present disclosure^βTub,Sxl/+; graph of survival curves for nos-Cas9/+ sterile males. Survival curves show the non-parametric maximum likelihood estimates (NPMLE) for the three male groups, as well as the 95% confidence intervals for the assisted (bootstrap) estimates with light shading, and representative non-uniqueness with dark shading. The y-axis shows the estimated percent survival. Both types of pgSIT males survived significantly longer than wild type males (P)<2.2^–16) Whereas no statistically significant difference was found between the two types of pgSIT males. Generalization of the Sun log rank test was used to test differences in survival curves.

FIG. 4E is a dgRNA as compared to a control w-male, in accordance with embodiments of the present disclosure^bTub,Sxl/+; table of survival data (longevity in days) for nos-Cas9/+ males.

Fig. 4F is a table of input parameters for an aedes aegypti population suppression model as disclosed herein, according to an embodiment of the present disclosure. All cited references as indicated in the tables are incorporated herein by reference in their entirety.

Fig. 4G is a graph of model predicted impact of release of pgSIT eggs (dark blue) on aedes aegypti mosquito population density compared to release of wolbachia-based Insect Incompatible Technology (IIT) (purple), release of insects carrying a dominant lethal gene (RIDL) (light blue), and female-specific RIDL (fsridl) (red), using an inhibition model as described herein, according to embodiments of the present disclosure. Release was performed weekly over a six month period, with release rates (relative to wild adults) as indicated in the legend. Model predictions were calculated using 2000 realizations of random implementation of the MGDrivE simulation framework for random mixing of 10,000 adult female aedes aegypti populations and the model parameters described in the table of fig. 4F. As shown, pgSIT release exceeds those of all other inhibition or reduction techniques, showing the greatest potential to eliminate local populations.

Fig. 4H shows a graph measuring sensitivity of the pgSIT model prediction to male mating competitiveness, life span reduction, with release rate of 200 eggs per wild adult, keeping all other parameters constant, as illustrated in the table of fig. 4F. Model predictions were calculated using 250 realizations of random implementation of the MGDrivE simulation framework for random mixing of 10,000 adult female aedes aegypti populations. According to embodiments of the present disclosure, as shown in the left panel, the release rate of 200 eggs per wild adult per week and elimination can be reliably achieved for 25% male mating competitiveness due to the reduction in longevity of the pgSIT construct remaining constant at 18%; however, 5% of male mating competitiveness cannot be eliminated, as is the case in RIDL adult males. According to embodiments of the present disclosure, as shown in the right panel, the release rate of 200 eggs per wild adult per week and keeping male mating competitiveness constant at 78% can reliably achieve elimination for a life reduction of less than or equal to 75%.

Fig. 4I is a graph showing a wide range of parameter values (concurrent variable life reduction and male mating competitiveness as indicated) for which local aedes aegypti eradication (tan tiles) can be reliably achieved, taking into account the 200 egg release rate per wild adult per week, according to embodiments of the present disclosure.

Fig. 4J shows a graph measuring sensitivity of the pgSIT model prediction to male mating competition, life span reduction, with release rates of 100 eggs per wild adult, keeping all other parameters constant, as illustrated in the table of fig. 4F. According to embodiments of the present disclosure, as shown in the left panel, the release rate of 100 eggs per wild adult per week and elimination can be reliably achieved for 50% male mating competitiveness due to the reduction in longevity of the pgSIT construct remaining constant at 18%; however, 25% of male mating competitiveness cannot be eliminated. According to embodiments of the present disclosure, as shown in the right panel, the release rate of 100 eggs per wild adult per week and keeping male mating competitiveness constant at 78%, elimination can be reliably achieved for a life reduction of less than or equal to 50%.

Fig. 4K is a graph showing a wide range of parameter values (concurrent variable life reduction and male mating competitiveness as indicated) for which local aedes aegypti eradication (tan tiles) can be reliably achieved, taking into account the release rate of 100 eggs per week for each wild adult.

Fig. 5 is a schematic diagram showing a factory located in the united states (blue point) producing pgSIT eggs for distribution (e.g., by drone) and release in remote areas around the world (e.g., south america, africa, and asia (pink point)), according to an embodiment of the present disclosure.

Detailed Description

Insect sterility technology (SIT) is an environmentally safe and proven technology for suppression to reduce wild populations. Embodiments of the present disclosure include methods of genetically modifying insects using CRISPR-based techniques, referred to herein as "precision-guided SIT" (pgSIT) methods. As disclosed in more detail throughout this disclosure, the pgSIT method relies mechanically on dominant genetic techniques that enable qualitative and concurrent or simultaneous qualitative and sterilization of insects. Concurrent or simultaneous characterization and sterilization of insect eggs allows for the release of the eggs into the environment ensuring the ability to render adult males sterile. For field applications, egg release eliminates the burden of manual characterization and sterilization of males, thereby reducing overall workload and improving scalability.

To demonstrate the efficacy of pgSIT technology according to embodiments of the present disclosure, multiple pgSIT systems were engineered and demonstrated with fruit flies as example insects. The genetic techniques and methods described and referenced herein should be understood to be applicable to a wide variety of insects.

The male qualitative pgSIT method and the male qualitative and sterile method disclosed herein use the precision and accuracy of CRISPR-based techniques to disrupt genes essential for female viability (for male characterization) or to disrupt genes essential for female viability and male fertility concurrently or simultaneously. The pgSIT method of the present disclosure utilizes a simple breeding scheme that requires two insect lines (a first parent line and a second parent line), one expressing an endonuclease (e.g., Cas9), and another expressing nucleic acid sequence construct having at least one guide polynucleotide that is targeted to one or more genes to be disrupted. A single mating between these two parental lines mechanically results in a simultaneous polynucleotide-directed (e.g., RNA-directed) dominant allele or dominant biallelic knockout of one or more target genes throughout development.

CRISPR technology refers to regularly interspaced clustered short palindromic repeats and has been extensively studied and modified for genome editing in most studied organisms, as disclosed in Sternberg and Doudna, mol. cell 58, 568-.

As used herein, with respect to CRISPR-based techniques, the term "guide polynucleotide" refers to a polynucleotide having a "synthetic sequence" capable of binding to a corresponding endonuclease protein (e.g., Cas9) and a variable target sequence (e.g., a nucleotide sequence found in an exon of a target gene) capable of binding to a genomic target. In some embodiments of the disclosure, the guide polynucleotide is a guide ribonucleic acid (gRNA). In some embodiments, the variable target sequence of the guide polynucleotide is any sequence within the target that is unique relative to the rest of the genome and is immediately adjacent to a protospacer sequence adjacent motif (PAM). The exact sequence of the PAM sequence may differ because different endonucleases require different PAM sequences. As used herein, the expression "single heterologous construct with two different single guide rnas (sgrnas)" refers to a double guide rna (dgrna).

With respect to the endonuclease proteins of CRISPR-based techniques, the term "endonuclease" refers to any suitable endonuclease protein or variant thereof that will be specifically directed by a selected guide polynucleotide to enzymatically knock out the target sequence of the guide polynucleotide. As used herein, the term "variant thereof" as used in reference to an endonuclease refers to any modified reference endonuclease in its enzyme functional form expressed in any suitable host organism or expression system and/or which includes enhancing the enzymatic activity of the endonuclease.

Examples disclosed throughout this disclosure represent methods for producing sterile male insect progeny in which both the female viability gene and the male fertility gene are disrupted using at least two guide polynucleotides. However, as will be understood by those of ordinary skill in the art, methods for directing male characterization include the presently disclosed methods in which genes essential for female viability are targeted, but genes for male sterility are not targeted. For example, referring to fig. 1A, an endonuclease parent insect (labeled Cas9 line) is crossed with a guide RNA parent (gRNA line) having two grnas (one blue, one green) targeting a female essential gene and a male sterile gene. However, for methods to direct male characterization, the gRNA line would not be genetically modified to express a male sterility gene.

As used herein, the term "integration" and similar terms refer to the introduction of a heterologous recombinant nucleic acid sequence into a target insect. As will be appreciated by those of ordinary skill in the art, techniques for insect Genetic modification are known and described, for example, in Cockburn et al, Biotechnology and Genetic Engineering Reviews, 2: 68-99, (1984), the entire contents of which are incorporated herein by reference. As used herein, integration can refer to integration of a recombinant nucleic acid sequence into the genome of a target insect. The genome of the target insect includes at least one chromosome of the target insect, but may include all relevant chromosomal copies. Thus, integration into the genome may be heterozygous or homozygous.

As used herein, the term "introducing an endonuclease into" a target insect refers to the recombinant introduction of an endonuclease into an insect such that the endonuclease is present in the insect. Introduction of the endonuclease into the insect does not require genomic integration, but may include genomic integration. For example, introduction of the endonuclease includes "depositing" the endonuclease into an insect, as described, for example, in Lin and Potter, G3, (2016), doi:10.1534/G3.116.034884, which is incorporated herein by reference in its entirety.

Additionally, although examples of the present disclosure include methods for obtaining up to 100% male sterile progeny, methods for producing less than 100% male sterile progeny are also included within the scope of the present disclosure. For example, in some embodiments of the present disclosure, a parent insect line expressing a guide polynucleotide may be heterozygous or homozygous for the guide polynucleotide (single, two or more guide polynucleotides). In some embodiments, the parent insect line expressing the guide polynucleotide is homozygous for the guide polynucleotide, thereby ensuring that all progeny receive the guide polynucleotide.

Referring to fig. 2A and 3A, in a further embodiment of the present disclosure, if the parent insect line expressing the endonuclease is male, the male parent may be heterozygous or homozygous for the endonuclease, and if the parent insect line expressing the endonuclease is female, the endonuclease may be deposited in the female, expressed heterozygously or homozygous.

Referring to fig. 2F, if two parental lines are homozygous for their respective endonuclease or guide polynucleotide, all or nearly all progeny receive the endonuclease and guide polynucleotide(s) due to non-mendelian complete penetrance. Thus, the desired phenotype in all offspring (e.g., all male insects or all male and sterile insects) can be produced in one generation. As used herein, the term "substantially all progeny" refers to at least 70%, 75% 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the progeny. Referring to table 3, in some embodiments of the present disclosure, the viability of the offspring is determined in adulthood.

In some embodiments of the disclosure, a method for directing male sex comprises introducing an endonuclease into a first insect parent and integrating at least one nucleic acid sequence construct into the genome (e.g., a plasmid vector) of a second insect parent, the nucleic acid sequence having at least one first guide polynucleotide (e.g., sgRNA or dgRNA) that targets a nucleotide sequence in a genomic sequence essential for females, and crossing the first insect parent and the second insect parent to produce all or substantially all male progeny. As used herein, the term "female essential genomic sequence" includes any genomic sequence or gene specific for a female insect. Examples of genomic sequences essential to females include sex-determining genes or female-specific splice variants thereof, genes or splice variants of genes not found in males, genes or splice variants of genes essential for female gonadal development, and/or genes or splice variants of genes not essential for male viability. Referring to FIG. 1B, non-limiting examples of genomic sequences essential for females include female-specific exons in sex-determining Drosophila genes Sxl, Tra, and Dsx, including homologs, orthologs, and paralogs thereof. As used herein, the term "homolog" refers to an comparable gene of an organism that confers the same function found in another organism. As used herein, the terms "ortholog" and "paralog" refer to the type of homolog. Orthologues are the corresponding genes in different lineages and are the result of speciation, and paralogues are produced by gene duplication.

Modulation and sex-specific alternative splicing mediated by the TRA protein or TRA/TRA-2 complex in insects is known and discussed in Pane et al, Development 129:3715-3725(2002), the entire contents of which are incorporated herein by reference. The male-and female-specificity of the splice product of the Dsx gene is known and discussed in Suzuki et al, Insect biochem mol Biol 31:1201-1211(2001), Salvemini et al, BMC Evol.biol.11,41(2011) and Scali et al, J.Exp.biol.208, 3701-3709 (2005), all of which are incorporated herein by reference in their entirety.

In some embodiments of the present disclosure, a method for directing male sex determination comprises introducing an endonuclease into a first insect parent and integrating at least one nucleic acid sequence construct into the genome of a second insect parent, the nucleic acid sequence having at least one first guide polynucleotide that targets a nucleotide sequence in a female essential genomic sequence selected from a female specific exon in a Tra and/or Dsx gene, including homologues, orthologs or paralogs thereof, wherein the first insect parent and the second insect parent are mated to produce all or substantially all male progeny.

In some embodiments of the present disclosure, a method for producing male sterile insect eggs includes introducing an endonuclease into a first insect parent and integrating at least one nucleic acid sequence construct into the genome of a second insect parent, the at least one nucleic acid sequence construct having at least one first guide polynucleotide targeting a female-essential genomic sequence necessary for female-specific viability or development and at least one second guide polynucleotide targeting a male-sterile genomic sequence necessary for male fertility, and crossing the first insect parent and the second insect parent to produce all or substantially all sterile male progeny. As used herein, the term "male sterile genomic sequence" refers to any male-specific genomic sequence essential for male fertility in insects that does not affect male insect development or male insect viability. Non-limiting examples of male-specific genomic sequences essential for male fertility in insects include the genes β Tubulin85D (β Tub), fuzzy onions (Fzo), protamine a (prota) and spermatocyte block (Sa) and homologues, orthologs and paralogs thereof. In some embodiments, the nucleic acid sequence construct comprises one or more second guide polynucleotides that target one or more male-specific genomic sequences necessary for male fertility. The functional conservation of β Tubulin85D, including the Anopheles and Aedes aegypti, is described in Catteruccia et al, nat. Biotechnol.23, 1414-1417 (2005) and Smith et al, inst mol.biol.16, 61-71 (2007), the entire contents of both of which are incorporated herein by reference.

As one of ordinary skill in the art will appreciate, many genes important for female viability and male fertility can be targeted. Additional female/male specific insect genes for disruption are discussed in Akbari et al, G33, 1493-1509 (2013) and Papa et al, (2016) doi:10.1101/081620, both of which are incorporated herein by reference in their entirety.

In some embodiments of the present disclosure, genetically modified insects and methods for generating genetically modified insects include insects from the order diptera, lepidoptera, or coleoptera.

In some embodiments of the present disclosure, genetically modified insects and methods for generating genetically modified insects include insects selected from mosquitoes of the genus superophelea, aedes, anopheles, or culex. Among these genera, example mosquito species include Aedes aegypti, Aedes albopictus, three rows of Moire subgenus (Ochrotatus trisriensis) (three rows of Aedes aegypti), Anopheles stephensi, Anopheles albopictus (Anopheles albicans), Anopheles gambiae, Anopheles quadratus Quadrifolia (Anopheles quadratus), Anopheles flooded (Anopheles freeborni), Culex species (Culex species), or Culex nigricans (Culiseta melanophore).

In addition, Cas 9-expressing strains have been developed in major dengue and malaria disease vectors including Aedes aegypti, Anopheles gambiae, and Anopheles stevensis, as described in Li et al, (2017) doi:10.1101/156778, Hammond et al, Nat.Biotechnol.34, 78-83 (2016) and Gantz et al, Proc.Natl.Acad.Sci.U.S.A.112, E6736-43 2015 (R), all of which are incorporated herein by reference in their entirety.

In some embodiments, the genetically modified insects and methods for generating genetically modified insects include any insect selected from any one of: fruit flies of the family of the Bacteroides family, selected from Mediterranean fruit flies (Medifly) (Ceratoisca chinensis), Mexfly (Anastrephea ludens), Oriental fruit flies (Oriental fruit flies) (Bactrocera dorsalis), Olive fruit flies (Olive fruit flies) (Bactrocera oleifera), Melon fruit flies (Mellon Fly) (Bactrocera cucurvatae), Natales fruit flies (Natal fruit flies) (Ceratoptera chinensis), Cherry fruit flies (Cherry fruit flies) (Rhagoguet blue), Qunsystem fruit flies (Bactrocera cereus), fruit flies (Cherry fruit flies) (Oriental fruit flies) (Haidophila), fruit flies (fruit flies) (Haidophila cera (Haidophila)), fruit flies (Queen fruit flies) (Bactrocera fruit flies) (Indian fruit flies) (Oriental fruit flies (Haidocera fruit flies), fruit flies (fruit flies) (Oriental fruit flies) (Haemata (Haidophyta)) and Haidophyta (Haidophyta) in Haidophyta (Haidophyceae), fruit flies (Haidophyta (Haidophys) and Haidophyta (Haidophyta) and Haidophysallow, Haidophyta) and Haidophyta (Haidophyta) including, New World Worm (New World Worm screw word) (trypanosoma japonicum (cochlioma hominivorax)), Old World Worm (Old World Worm screw word) (chrysomyia fascicularis (chrysomyia subsira)), Australian sheep green-head/green fly (austrian sheep yellow/green fly) (Lucilia cuprina), pink bollworm (ping bollworm) (pink bollworm (pecnophora gossypiella), European Gypsy moth (Lymantria mollis)), Orange Worm (the naval Orange Worm) (Orange Borer (amyristella), wheat Worm (Peach kernel) (ostrinia), pink moth (pink bollworm) (ostrinia nubilalis), pink moth (ostrinia nubilalis), black rice stem (ostrinia nubilalis) (ostrinia), black rice stem Borer (ostrinia nubila nubilalis) (ostrinia nubilalis), black rice stem Borer (ostrinia nubila (ostrinia), black rice stem (ostrinia nubila (ostrinia), black moth (ostrinia nubila (ostrinia) and (ostrinia nubila (ostrinia nubila) and (ostrinia nubila, Cotton Boll weevil (Boll Weeviil) (Cotton Boll weevil (Anthonomous grandis)), potato beetle (Phoma potato beetle (Leptinotarsa decemlineata)), grape mealybug (vitamin mealybug) (Ficus carica (Planococcus ficus)), Asian citrus psylla (Asian citrus psylla) (diaphorina citri), Spotted fruit fly (Spotted fruit fly (drosophila), blue green cicada (Bluegreen sharpsophilter) (Graphophaia grandis (apple budra)), glass leaf worm (glass leaf moth) (glass leaf moth (apple budworm)) and Brown apple budworm (apple budworm moth (apple budworm), apple leaf moth (apple leaf moth) (apple budworm moth (apple budworm), apple leaf moth (apple leaf moth), Brown apple leaf moth (apple leaf moth), apple leaf moth (Brown apple leaf moth), apple leaf moth (apple leaf moth), apple leaf moth (Brown apple leaf moth (apple leaf moth), apple leaf moth (Brown leaf moth), apple leaf moth (Brown apple leaf moth), apple leaf moth (Brown leaf moth (apple leaf moth), apple leaf moth (Brown leaf moth (apple leaf moth), grape leaf moth (, Asian longicorn beetles (Aspian longicorn beetles) (Anoplophora glabipinnis), Cocotilus Rhinoceros beetles (Coconut Rhinoceros beetles) (Coconut Moth (Oryces rhynchophoros)), Ceriporious chinensis (Emerad Ash Moth) (Ceriporio chinensis (Agrilus spicata)), European grape Moth (European Gypsy Moth) (Lobesia carthamiana (Lobesia bornana)), European Gephysalis (Lymantria dispar), Pseudoapple leaf Moth (Fable Codlmortis) (apple Isotibialis (Thauotia leucotricha)), fire ants (selected from Solenopsis Burserra Buren (Solenopsis Buren) and Pyrenopsis glauca (S. richarda), and African yellow corn beetles (Helicoverum), yellow corn Borer (Ostrinia punctifera (Ostrinia) and African yellow corn Borer (Osbeckia), yellow corn Borer (Ostrinia) and yellow corn Borer (Osmanthus (Osbeckia), African yellow corn Borer (Osmanthus) insect (Osbeckia) Western corn rootworm (Western rootworm) (corn rootworm (diabrotica virgifera)), Whitefly (Whitefly) (bemisia tabaci)), HouseFly (HouseFly) (Musca domestica), Green Fly (Green Bottle Fly) (Lucilia cuprina), silkworm Moth (Silk Moth) (silkworm (Bombyx mori)), Red mussel (Red Scale) (Aonidiella aurantia), heartworm (Dog heartworm) (heartworm (diaperi), southern pine maggot (southern bean Fly) (saprorhiza large (delbrueckia), Western yellow Fly (blowfly), and some species of trypanosoma (trypanosoma), cotton Fly (blowfly), and (blowfly, or trypanosoma melanogaster Fly (blowfly) Pisca (Warble Fly) (selected from the group consisting of Dermata bovis (Hypoderma bovis) or Dermata striatus (Hypoderma Lineatum)), Ceratoxylum maculatum (Spotted Lantern. flavus) (Lycrma deltica), Pectinatus punctatus (Khaprabeetle) (Trogopteroderma granarium), Melissae mellitus (Honeybee mite (varroa destructor)), Termite (Coptotermes formosanus), Phycomyces ferrugineus (Hemlocofelus formosanus) (Hemlock Woorganism), Phyllophora ferrugineus (Hemlocontrophora), Phyllophora punctatus (Waldens) Miyau (Wallichen), Phyllophora regia carotovora (Waldonova), Phyllophora juglandica (Pillla punctatus (Wallichia), Phyllophora punctata (Waldens punctatus) (Pillla punctatus (Thunberg), Phyllophora punctatus (Pillla punctatus) (Pillla punctata), Phyllophora punctatus (Pilitura), Pillla punctatus (Pilitura), Pilitura punctatus (Pilitura) Pimenta punctatus (Pimenta punctatus) (Pilitura), Pilitura punctatus (Pilitura) Piercus) Pimenta punctatus (Piperi) Pilitura), Piperi punctatus (Pilitura), Pilitura) Piercus (Pilitura) Piperi punctatus (Pilitura), Piperi (Pimpinus punct, Red flower beetle (Red flower beetle) (tribolium castaneum), Green peach Aphid (Green peach Aphid) (Myzus persicae), Cotton Aphid (Cotton aphi) (Cotton Aphid (aphis gossypii)), Brown planthopper (Brown plant louse) (nilaparvata lugens), Beet armyworm (Beet armyworm) (spodoptera exigua)), Western flower Thrips (Western flower Thrips) (frankliniella occidentalis), apple budworm (Codling moth) (cydia pomonella), Pea weevil (Cowpea weevil) (Green flower), Pea louse (Pea Aphid)), Pea louse (Pea Aphid), Pea louse (Pea Aphid) (Green flower), Pea louse (Pea Aphid (Pea), Pea louse (Pea Aphid) (Pea Aphid (Pea), Pea Aphid (Pea), Cotton bollworm) (Pea Aphid (Pea), Cotton boll (Pea Aphid (Green), Cotton boll (Pea Aphid (Pea) and Pea Aphid (Green), Cotton bollworm) (Green beetle) are included).

In some embodiments of the disclosure, suitable endonucleases include a CRISPR-associated sequence 9(Cas9) endonuclease or variant thereof, a CRISPR-associated sequence 13(Cas13) endonuclease or variant thereof, a CRISPR-associated sequence 6(Cas6) endonuclease or variant thereof, a CRISPR (Cpf1) endonuclease or variant thereof from prevotella and francisella 1, or a CRISPR (Cms1) endonuclease or variant thereof from microgenerates and smithlla 1.

In some embodiments of the disclosure, suitable endonucleases include streptococcus pyogenes Cas9(SpCas9), staphylococcus aureus Cas9(SaCas9), francisella noveniculans Cas9(FnCas9), or variants thereof. Variants may include a Protospacer Adjacent Motif (PAM) SpCas9(xCas9), high fidelity SpCas9(SpCas9-HF1), high fidelity SaCas9, or high fidelity FnCas 9.

In other embodiments of the disclosure, the endonuclease comprises a Cas fusion nuclease comprising a Cas9 protein or variant thereof fused to a fokl nuclease or variant thereof. Variants of the Cas9 protein of the fusion nuclease include catalytically inactive Cas9 (e.g., dead Cas 9).

In some embodiments of the present disclosure, the endonuclease may be a Cas9, Cas13, Cas6, Cpf1, CMS1 protein, or any variant thereof, derived from or expressed by: methanococcus maripalensis C7, Corynebacterium diphtheriae, Corynebacterium hyperboloides YS-314, Corynebacterium glutamicum (ATCC 13032), Corynebacterium glutamicum R, Corynebacterium chrysogenum (Corynebacterium kpppenstetii) (DSM 44385), Mycobacterium abscessus (ATCC19977), Nocardia gangeticus (Nocardia farcina) IFM10152, Rhodococcus erythropolis (Rhodococcus erythropolis) PR4, Rhodococcus giardia (Rhodococcus jositii) RHA 2, Rhodococcus opaque (Rhodococcus rhodochrous) B4(uid36573), Thermomyces cellulolyticus (Acidorhizophilus cellulolyticus)11B, Arthrobacter chlorophenol (Arthrobacter rhodochrophilus) A6, Kribella flavipetalaria (Corynebacterium thermoacidophilus) DSM 17836, Corynebacterium thermoacidophilum 433176, Bifidobacterium thermobacter xylinum (Bifidobacterium longum) DSM 6776, Bifidobacterium longum (Corynebacterium thermobacter xylinum) DSM 6776, Bifidobacterium longum (Bifidobacterium longum 433638776), Bifidobacterium longum (Bifidobacterium longum) DSM 3638776), Bifidobacterium longum 433676, Bifidobacterium longum (Bifidobacterium longum) (Bifidobacterium sp) DSM 3638776, Bifidobacterium longum 4335, Bifidobacterium longum (Bifidobacterium longum) and Bifidobacterium longum (Bifidobacterium longum) Lactobacillus acidophilum 3676, Bifidobacterium longum 3676, Bifidobacterium sp Flavobacterium psychrophilum JIP 0286, Excherisca muciniphila (Akkermansia muciniphila) (ATCC BAA 835), Rosa photosynthetic (Roseaus casetenholzii) (DSM 13941), Rosa rugosa (Roseaflex) RS1, Synechocystis PCC 03, Trachelospermum micans (Elusiobium miniatum) Pei191, uncultured Termite group 1 (uncultured Termite group 1 bacterial species) Rs D17, filamentous Bacillus succinogenes (Fibrobacter succinogenes) S85, Bacillus cereus (ATCC 10987), Streptococcus innocuus (Listeria nococcua), Lactobacillus casei (Labaceri) casei, Lactobacillus rhamnosus, Lactobacillus casei C118, Streptococcus salivarius (Streptococcus faecalis) S1, Streptococcus agalactis S1, Streptococcus agalactia Streptococcus agalactiae S1, Streptococcus agalactia Streptococcus faecalis strain 1, Streptococcus faecalis strain CGI 1, Streptococcus faecalis strain (Streptococcus faecalis strain) Streptococcus faecalis strain, Streptococcus mutans NN2025(uid46353), Streptococcus mutans, Streptococcus pyogenes (Streptococcus pyogenes) M1 GAS, Streptococcus pyogenes MGAS5005, Streptococcus pyogenes MGAS2096, Streptococcus pyogenes MGAS9429, Streptococcus pyogenes MGAS10270, Streptococcus pyogenes MGAS6180, Streptococcus pyogenes MGAS315, Streptococcus pyogenes SSI-1, Streptococcus pyogenes MGAS10750, Streptococcus pyogenes NZ131, Streptococcus thermophilus CNRZ1066, Streptococcus thermophilus LMD-9, Streptococcus thermophilus LMG 18311, Clostridium botulinum A3 Loch Maree, Clostridium botulinum B Ekludu nd17B, Clostridium botulinum Ba 4657, Clostridium botulinum F Langeland, Clostridium cellulolyticum H10, Fenugera fungus (Finegogonia) (ATCC 29328), Clostridium procytococcus (Eubacterium tarum) ATCC33656), Mycoplasma mobilis (Mycoplasma mobilis Mycoplasma pneumoniae), Mycoplasma pneumoniae (Mycoplasma pneumoniae) Mycoplasma pneumoniae (Mycoplasma mobilis Mycoplasma pneumoniae (Mycoplasma pneumoniae) M3553), Mycoplasma mobilis strain (Mycoplasma pneumoniae, Mycoplasma pneumoniae (Mycoplasma pneumoniae) Mycoplasma pneumoniae (Mycoplasma pneumoniae, streptococcus moniliforme (Streptococcus moniliformis) (DSM12112), Bradyrhizobium lenticulmor (Bradyrhizobium) BTai1, Nitrobacter hancei (Nitrobacter hamburgensis) X14, Rhodopseudomonas palustris (Rhodopseudomonas palustris B18), Rhodopseudomonas palustris (B5), Microbacterium parvulus (Paracoccus lavamentivorans) DS-1, Rhodobacter xylinum (Dinospora shibae) DFL 12, Acetobacter diazotrophicus (Gluconobacter diazotrophoreicus) Pal 5FAPERJ, Acetobacter diazotrophicus Pal 5JGI, Azospirillum azotobacter B510(uid 85), Rhodospirillum rubrum (Neisseria rubra) (ATCC 70), Acidobacter paracasei (Corynebacterium paracasei), Campylobacter xylinum roseum (Corynebacterium parvurica) Pal 5 JN, Campylobacter xylinum vulgare (Campylobacter xylinum) Hayata, Campylobacter xylinus strain ATCC 2938, Campylobacter xylinus meningitidis 8191, Campylobacter xylinus cerealis (Campylobacter asiaticum) Hayata, Vibrio-3, Vibrio-Haemarrhizia lactis (Caminus) Hayata, Vibrio-3, Vibrio-2, Vibrio-Hayata, Vibrio-3, Vibrio-Hayata, Vibrio-3, Vibrio-la parahaemophilus, Vibrio-la, Campylobacter jejuni, Campylobacter rhodobacter (Campylobacter) RM2100, Helicobacter (Helicobacter hepaticus), Wollaromyces succinogenes (Wolinella auccinogens), Tolulomonas solani (Tolumonas auensis) DSM 9187, Pseudoalteromonas maxima (Pseudoalteromonas amylotica) T6c, Shewanella peleana (ATCC 700345), Legionella pneumophila (Legionella pulcherhia Paris), Actinobacillus succinogenes (Actinobacillus succinogenes)130Z, Pasteurella multocida (Pasteurella histolytica), Francisella novensis novvida 63vida U112, Francisella tularenaria, Francisella tularensis, FSislea farrella 198, Francisella calophyllaria, ATCC 34135, W34135 or Wolomycelia fargeisseria.

Referring to fig. 4A-4C, the mating competitiveness of sterile males produced using the pgSIT method according to embodiments of the present disclosure indicates that these sterile males can successfully mate and compete successfully for female partners in the field. 4D-4E, the life spaces (lifespaces) of sterile males produced using the pgSIT method according to embodiments of the present disclosure indicate that the longevity (total number of days) of these sterile males is at least as long as, if not longer than, the longevity of corresponding wild-type males. Mating competitiveness and longevity are major factors in achieving local elimination, as larval resources are abundant once initial inhibition or reduction is achieved, and thus less affected by greater consumption of released immature forms. Egg release results in rapid population suppression or reduction from the outset, as the hatched larvae consume resources that would otherwise be available to the fertile larvae. Furthermore, pgSIT male-sterile eggs according to embodiments of the present disclosure may produce hatched larvae when released in the field, as female lethality occurs at the embryo/larval stage, resulting in maximal consumption of larval resources by releasing immature forms.

In addition, the pgSIT method disclosed herein does not rely on chromosomal translocations, chemical sterilants, radiation, antibiotics, or bacterial infections, which can severely compromise the health and mating competitiveness of the released sterile males.

Referring to fig. 4F-4K, using the MGDrivE simulation framework described in example 5 herein, 200pgSIT male sterile eggs were simulated weekly release from each wild adult, demonstrating that a wide range of parameter values for local aedes aegypti eradication were reliably achieved.

Referring to fig. 5, a method of inhibiting or reducing an insect population of an insect species including a disease vector and an agricultural pest includes introducing male-sterile eggs produced using the pgSIT method of the present disclosure into an area in need of targeted insect inhibition or reduction.

Some embodiments of the disclosure include the development of a breeding facility to separately propagate homozygous endonucleases (e.g., Cas9) and lines expressing dgrnas. In some embodiments, automated work flows are implemented to sex sort immature stages (e.g., Cas9 female versus dgRNA male) and combine into cages for maturation, mating, and breeding of eggs. Sex sorting can be accomplished by a number of suitable means, including mechanical size separation, automated copas sex sorting platform for combinatorial genetic profiling (Union Biometrica), or automated robotic optical sorting. Suitable methods for sex sorting are discussed in Papathanoss et al, Transgenic entities: techniques and applications 83-100, (October,2014) and Gilles et al, Acta trop.132, S178-S187 (2014), the entire contents of which are incorporated herein by reference.

In some embodiments, the pgSIT method to produce sterile male eggs is particularly effective for insect species diapauzing during the egg phase. Insects that diapauze during the egg phase include, for example, aedes aegypti and aedes albopictus, as described in Diniz et al, parasit.vectors 10,310(2017), the entire contents of which are incorporated herein by reference. This diapause will accumulate expandable (scalable) eggs for excess (interstitial) release. Thus, as generally depicted in FIG. 5, a single efficient pgSIT egg production facility can distribute pgSIT eggs to many outlying fields around the world, where they can be simply hatched, raised and released, eliminating or reducing the logistical burden of sex sorting, sterilization and releasing fragile adult males at each field site, thereby improving scalability and efficiency, enabling broader large-scale population suppression or reduction capabilities.

The following examples are given for illustrative purposes only and do not limit the scope or content of the present application.