US-12622419-B2 - Endonuclease sexing and sterilization in insects

Abstract

Methods of the disclosed precision guided sterile insect technique (pgSIT) include methods for directing male sexing in a genetically modified insect and methods of producing a progeny of genetically modified sterile male insect egg. These methods include integrating at least one nucleic acid sequence into a genome of a first insect, the at least one nucleic acid sequence having at least one first guide polynucleotide targeting a female-essential genomic sequence that is required for female-specific viability, introducing an endonuclease into a second 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. For male sterility a second guide polynucleotide targets a male sterility genomic sequence that is required for male-specific sterility.

Inventors

• Nikolay P. Kandul
• Omar S. Akbari

Assignees

• THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Dates

Publication Date

20260512

Application Date

20181119

Claims (20)

1. 1 . A method of directing male sexing in a progeny of genetically modified insects, the method comprising: (a) providing a first insect strain, wherein the first insect strain has integrated into its genome: (i) at least one nucleic acid sequence comprising at least one first guide polynucleotide targeting a female-essential genomic sequence that is required for female-specific viability of the first insect strain, wherein the female-essential genomic sequence is a gene or a splice-variant of a gene selected from the group consisting of sex lethal (Sxl), transformer (Tra), and doublesex (Dsx), and (ii) at least one nucleic acid sequence comprising at least one second guide polynucleotide targeting a male sterility genomic sequence that is required for male-specific fertility of the first insect strain, wherein the male sterility genomic sequence is a βTubulin 85D (βTub) gene; (b) providing a second insect strain, wherein the second insect strain has integrated into its genome a nucleic acid sequence encoding an endonuclease, and wherein: the first insect strain and the second insect strain are the same insect species, and the endonuclease is directed by the first and second guide polynucleotides to enzymatically knock-out the female-essential genomic sequence and the male sterility genomic sequence, respectively, when present in the same insect; and (c) genetically crossing the first insect strain and the second insect strain, thereby producing a progeny of genetically modified insects comprising the endonuclease and the at least one nucleic acid sequence, wherein the progeny comprises sterile male insect eggs or sterile male insects.
2. 2 . The method of claim 1 , wherein the progeny of genetically modified insects produced in (c) comprises sterile male insect eggs.
3. 3 . The method of claim 1 , wherein the nucleic acid sequences comprising the at least first and second guide polynucleotides are integrated into the genome of the first insect strain by homozygous integration into all chromosome copies in the genome.
4. 4 . The method of claim 1 , wherein the nucleic acid sequences comprising the at least first and second guide polynucleotides are introduced into the first insect during an embryonic stage.
5. 5 . The method of claim 1 , 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. 6 . The method of claim 1 , wherein the at least one first guide polynucleotide comprises more than one first guide polynucleotide, each of which targets a different region of the same female-essential genomic sequence that is required for female-specific viability.
7. 7 . The method of claim 1 , wherein the at least one first guide polynucleotide comprises more than one first guide polynucleotide, each of which targets a different female-essential genomic sequence that is required for female-specific viability.
8. 8 . The method of claim 1 , wherein the at least one first guide polynucleotide comprises more than one first guide polynucleotide, each of which targets a different gene selected from the group consisting of: sex lethal (Sxl), transformer (Tra), and doublesex (Dsx).
9. 9 . The method of claim 8 , wherein the more than one first guide polynucleotide comprises two first guide polynucleotides, each of which targets a different gene selected from the group consisting of: Sxl, Tra, and Dsx.
10. 10 . The method of claim 8 , wherein the more than one first guide polynucleotide comprises two first guide polynucleotides, each of which targets a different gene selected from the group consisting of: Sxl and Dsx.
11. 11 . The method of claim 1 , wherein: when the second insect is a male, the nucleic acid sequence encoding the endonuclease is integrated into the genome of the second insect by homozygously integrating a gene encoding the endonuclease, and when the second insect is a female, the nucleic acid sequence encoding the endonuclease is integrated into the genome of the second insect by homozygously or heterozygously integrating a gene encoding the endonuclease or by depositing an endonuclease protein into the second insect.
12. 12 . The method of claim 1 , wherein the nucleic acid sequence encoding the endonuclease is integrated into the genome of the second insect during an embryonic stage.
13. 13 . The method of claim 1 , 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, CRISPR-associated sequence 6 (Cas6) endonuclease or a variant thereof, a CRISPR from Prevotella and Francisella 1 (Cpf1) endonuclease or a variant thereof, or a CRISPR from Microgenomates and Smithella 1 (Cms1) endonuclease, or a variant thereof; a Streptococcus pyogenes Cas9 (SpCas9), a Staphylococcus aureus Cas9 (SaCas9), a Francisella novicida Cas9 (FnCas9), or a variant thereof; a Cas fusion nuclease comprising a Cas9 protein or a variant thereof fused with a FokI nuclease or variant thereof; or a Cas9, Cas13, Cas6, Cpf1, Cms1 protein or any variant thereof derived from Methanococcus maripaludis C7, Corynebacterium diphtheria, Corynebacterium efficiens YS-314, Corynebacterium glutamicum (ATCC 13032), Corynebacterium glutamicum (ATCC 13032), Corynebacterium glutamicum R, Corynebacterium kroppenstedtii (DSM 44385), Mycobacterium abscessus (ATCC 19977), Nocardia farcinica IFM10152, Rhodococcus erythropolis PR4, Rhodococcus jostii RHA1, Rhodococcus opacus B4 (uid36573), Acidothermus cellulolyticus 11B, Arthrobacter chlorophenolicus A6, Kribbella flavida (DSM 17836, uid43465), Thermomonospora curvata (DSM43183), Bifidobacterium dentium Bd1, Bifidobacterium longum DJO10A, Slackia heliotrinireducens (DSM 20476), Persephonella marina EX H1, Bacteroides fragilis NCTC 9434 , Capnocytophaga ochracea (DSM 7271), Flavobacterium psychrophilum JIP02 86 , Akkermansia muciniphila (ATCC BAA 835), Roseiflexus castenholzii (DSM 13941), Roseiflexus RS1, Synechocystis PCC6803 , Elusimicrobium minutum Pei191, uncultured Termite group 1 bacterium phylotype Rs D17 , Fibrobacter 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 dysgalactiae equisimilis GGS 124, Streptococcus equi zooepidemicus MGCS10565, Streptococcus gallolyticus UCN34 (uid46061), Streptococcus gordonii Challis subst CH1, Streptococcus mutans NN2025 (uid46353), Streptococcus mutans, 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 thermophiles CNRZ1066, Streptococcus thermophiles LMD-9, Streptococcus thermophiles LMG 18311, Clostridium botulinum A3 Loch Maree, Clostridium botulinum B Eklund 17B, Clostridium botulinum Ba4 657, Clostridium botulinum F Langeland, Clostridium cellulolyticum H10, Finegoldia magna (ATCC 29328), Eubacterium rectale (ATCC 33656), Mycoplasma gallisepticum, Mycoplasma mobile 163K, Mycoplasma penetrans, Mycoplasma synoviae 53, Streptobacillus moniliformis (DSM 12112), Bradyrhizobium BTAi1 , Nitrobacter hamburgensis X14, Rhodopseudomonas palustris BisB18, Rhodopseudomonas palustris BisB5 , Parvibaculum lavamentivorans DS-1 , Dinoroseobacter shibae . DFL 12, Gluconacetobacter diazotrophicus Pal 5 FAPERJ, Gluconacetobacter diazotrophicus Pal 5 JGI, Azospirillum B510 (uid46085), Rhodospirillum rubrum (ATCC 11170), Diaphorobacter TPSY (uid29975), Verminephrobacter eiseniae EF01-2, Neisseria meningitides 053442, Neisseria meningitides alpha14, Neisseria meningitides Z2491, Desulfovibrio salexigens DSM 2638, Campylobacter jejuni doylei 269 97, Campylobacter jejuni 81116, Campylobacter jejuni, Campylobacter lari RM2100, Helicobacter hepaticus, Wolinella succinogenes, Tolumonas auensis DSM 9187, Pseudoalteromonas atlantica T6c, Shewanella pealeana (ATCC 700345), Legionella pneumophila Paris, Actinobacillus succinogenes 130Z, Pasteurella multocida, Francisella tularensis novicida U112, Francisella tularensis holarctica, Francisella tularensis FSC 198, Francisella tularensis tularensis, Francisella tularensis WY96-3418, or Treponema denticola (ATCC 35405).
14. 14 . The method of claim 1 , wherein the first insect strain and the second insect strain are species in an Order selected from the group consisting of: Diptera, Lepidoptera, and Coleoptera.
15. 15 . The method of claim 14 , wherein: the first and second insect strains are a mosquito species from the genera Stegomyia, Aedes, Anopheles , or Culex ; or the first and second insect strains are a species selected from the group consisting of: 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 decemlineata ), the vine mealybug ( Planococcus ficus ), Asian citrus psyllid ( Diaphorina citri ), Spotted wing drosophila ( Drosophila suzukii ), Bluegreen sharpshooter ( Graphocephala atropunctata ), Glassy winged sharpshooter ( Homalodisca 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 longhorned 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 ( Helicoverpa armigera ), Spotted Lanternfly ( Lycorma delicatula ), Africanized honeybee ( Apis mellifera scutellata ), Fruit and shoot borer ( Leucinodes orbonalis ), corn root worm ( Diabrotica spp.), Western corn rootworm ( Diabrotica virgifera ), Whitefly ( Bemisia tabaci ), House 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 ), Horse Fly ( Tabanus sulcifrons ), Horn Fly ( Haematobia irritans ), Screwworm Fly selected from Cochliomyia macellaria ( C. macellaria ), C. hominivorax, C. aldrichi , or C. minima , Tsetse Fly ( Glossina spp.), Warble Fly selected from Hypoderma bovis or Hypoderma lineatum , Spotted lanternfly ( 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 ), and Cotton bollworm ( Helicoverpa armigera ).
16. 16 . A progeny of insect eggs produced by the method of claim 1 , wherein the progeny's genome comprises: (a) at least one nucleic acid sequence comprising at least one first guide polynucleotide targeting a female-essential genomic sequence that is required for female-specific viability, wherein the female-essential genomic sequence is a gene or a splice-variant of a gene selected from the group consisting of sex lethal (Sxl), transformer (Tra), and doublesex (Dsx); (b) at least one nucleic acid sequence comprising at least one second guide polynucleotide targeting a male sterility genomic sequence that is required for male-specific fertility, wherein the male sterility genomic sequence is a βTubulin 85D (βTub) gene; and (c) a nucleic acid sequence encoding an endonuclease, wherein the endonuclease is directed by the first and second guide polynucleotides to enzymatically knock-out the female-essential genomic sequence and the male sterility genomic sequence, respectively, when present in the same insect.
17. 17 . A genetically modified sterile male insect whose genome comprises: (a) at least one nucleic acid sequence comprising at least one first guide polynucleotide targeting a female-essential genomic sequence that is required for female-specific viability, wherein the female-essential genomic sequence is a gene or a splice-variant of a gene selected from the group consisting of sex lethal (Sxl), transformer (Tra), and doublesex (Dsx); (b) at least one nucleic acid sequence comprising at least one second guide polynucleotide targeting a male sterility genomic sequence that is required for male-specific fertility, wherein the male sterility genomic sequence is a βTubulin 85D (βTub) gene; and (c) a nucleic acid sequence encoding an endonuclease, wherein the endonuclease is directed by the first and second guide polynucleotides to enzymatically knock-out the female-essential genomic sequence and the male sterility genomic sequence, respectively, in the genome of the genetically modified sterile male insect, and wherein the genetically modified sterile male insect is capable of increasing the rate of unhatched eggs by mating with wild-type female insects.
18. 18 . The method of claim 13 , wherein the variant of SpCas9, SaCas9, or FnCas9 comprises a protospacer adjacent motif (PAM) SpCas9 (xCas9), high fidelity SpCas9 (SpCas9-HF1), a high fidelity SaCas9, or a high fidelity FnCas9.
19. 19 . The method of claim 13 , wherein the variant of the Cas9 protein fused with the FokI nuclease or variant thereof comprises a catalytically inactive Cas9 (dead Cas9).
20. 20 . The method of claim 15 , wherein the mosquito species is selected from the group consisting of: Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus ( Aedes triseriatus ), Anopheles stephensi, Anopheles albimanus, Anopheles gambiae, Anopheles quadrimaculatus, Anopheles freeborni, Culex species , and Culiseta melanura.

Description

CROSS-REFERENCE TO RELATED APPLICATION The present application claims priority to and the benefit of U.S. Provisional Application Ser. No. 62/589,405 filed on Nov. 21, 2017, entitled “NOVEL STERILE INSECT TECHNIQUE,” the entire content of which is incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No(s). 5K22AI113060 and 1R21AI123937 awarded by the National Institutes of Health and Grant No: HR0011-17-2-0047 awarded by the Defense Advanced Research Project Agency. The government has certain rights in the invention. BACKGROUND Mass-production and release of sterile males, known as the Sterile Insect Technique (SIT), has historically been used to control, and eradicate, insect pest populations dating back to the mid-1930s. Previous methodologies have relied on DNA-damaging agents for sterilization, substantially reducing overall fitness and mating competitiveness of released males. To overcome these issues, microbe-mediated infertility techniques such as Wolbachia-based incompatible insect technique (IIT) and modern genetic SIT-like systems such as the Release of Insects carrying a Dominant Lethal (RIDL), and other methodologies to release fertile males that genetically kill females such as female-specific RIDL (fsRIDL), and autosomal-linked X-chromosome shredders have been developed. While these first-generation genetic SIT technologies represent significant advances, IIT strictly requires no infected females to be released which is difficult to achieve in the field, and the use of tetracycline known to ablate the microbiota compromises the fitness of RIDL/fsRIDL males, and X-chromosome shredders can in principle only be developed in species with heterogametic sex chromosomes, thereby limiting wide applicability to other species. Therefore, it would be logistically advantageous to employ more efficient SIT-based technologies that can be deployed as eggs by which only sterile males would survive. SUMMARY Aspects of embodiments of the present disclosure are directed to methods including precision guided sterile insect technique (pgSIT). In some embodiments of the present disclosure, a method of directing male sexing in a genetically modified insect includes: integrating at least one nucleic acid sequence into a genome of a first insect, the at least one nucleic acid sequence having at least one first guide polynucleotide targeting a female-essential genomic sequence that is required for female-specific viability; introducing an endonuclease into a second insect, the second insect capable of being genetically crossed with 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 male insect eggs mature to adulthood. In some embodiments of the present disclosure, a method of producing a progeny of genetically modified sterile male insect eggs includes: integrating at least one nucleic acid sequence into a genome of a first insect, the at least one nucleic acid sequence having at least one first guide polynucleotide targeting a female-essential genomic sequence that is required for female-specific viability; introducing an endonuclease into a second insect, the second insect capable of being genetically crossed with the first insect, wherein the at least one nucleic acid sequence further includes at least one second guide polynucleotide targeting a male sterility genomic sequence that is required for male-specific sterility; and genetically crossing the first insect and the second insect to produce a progeny of genetically modified sterile male insect eggs. In some embodiments of the present disclosure, the integrating at least one nucleic acid sequence into the genome of the first insect includes homozygous integration into all chromosome copies in the genome. In some embodiments, the integrating the at least one nucleic acid sequence includes introducing the at least one nucleic acid sequence into the first insect during an 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 include at least one guide ribonucleic acid (gRNA). In some embodiments of the present disclosure, the female-essential genomic sequence includes a gene essential for female-specific viability or a female-specific exon essential for female-specific development and/or female-specific viability. In some embodiments of the present disclosure, the at least one first guide polynucleotide includes more than one first guide polynucleotide each of which targets a different region of the same female-essential genomic sequence that is required for female-specific viability. In some embodiments of the present disclosure, the at least one first guide polynucleotide includes more than one first guide polynucleo