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Endonuclease sexing and sterilization in insects

An endonuclease and insect technology, applied in the direction of nucleic acid vectors, hydrolytic enzymes, biochemical equipment and methods, etc., can solve the problem of limiting the wide applicability of other species, unable to release infected females, and damaging the health of RIDL/fsRIDL males And other issues

Pending Publication Date: 2020-08-25
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While these first generation genetic SIT technologies represent a significant advance, IITs strictly require that infected females not be released, which is difficult to achieve in the field, and the use of tetracyclines to ablate the microbiota is known to impair the fitness of RIDL / fsRIDL males , and the X chromosome shredder can in principle only be developed in species with heterogametic chromosomes, limiting broad applicability to other species

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  • Endonuclease sexing and sterilization in insects
  • Endonuclease sexing and sterilization in insects
  • Endonuclease sexing and sterilization in insects

Examples

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Embodiment 1

[0099] Example 1. Binary CRISPR-induced female masculinization / lethality or male sterility. To engineer pgSIT, single guide RNA (sgRNA) and spCas9 (from here Cas9) expression lines were generated in Drosophila. A total of 9 homozygous sgRNA lines were developed to target genes essential for female viability or genes important for male fertility. For female viability, these genes include sex-specific alternatively spliced ​​sex-determining genes, which include eg Figures 1B-1C As shown in and in Slee and Bownes, Q. Rev. Biol. 65, 175-204 (1990); Bell et al., Cell 65, 229-239 (1991); Boggs et al., Cell 50, 739-747 (1987) and Burtis and Baker , the sex-determining lethal gene (Sxl, two separate transgenic lines-sgRNA Sxl , sgRNA Sxl-B ), transforming gene (tra, two separate lines-sgRNA Tra , sgRNA Tra-B ) or bisex genes (dsxF, sgRNA DsxF ), the entire contents of all documents are incorporated herein by reference. To disrupt male fertility, target genes that are active d...

Embodiment 2

[0101] Example 2. Create populations of up to 100% sterile males. In some embodiments of the present disclosure, the disclosed pgSIT methods can be used to disrupt genes essential for female viability and / or male sterility. In some embodiments, the disclosed pgSIT method can be used to concurrently or simultaneously disrupt genes necessary for female viability and male sterility to convert most or all (up to 100%) of surviving F 1 Offspring are genetically directed to sterile males. To achieve this directed characterization and sterility, three additional homozygous lines expressing multiple double gRNA (dgRNA) combinations including dgRNA βTub,Sxl , dgRNA βTub,Tra and dgRNA βTub,DsxF ,like Figure 1C shown in . To genetically assess the activity of these pgSIT lines, each line was bidirectionally crossed with wild-type or homozygous Cas9 (nos-Cas9, vas-Cas9, or Ubi-Cas9). refer to Figure 2A-2B , wild-type crosses did not produce significant sex bias or impaired ferti...

Embodiment 3

[0104] Example 3. Full penetrance due to zygotic expression . Maternal deposition of the Cas9 / gRNA complex in developing embryos is sufficient to ensure non-Mendelian inheritance of mutations in receiving offspring, even if those offspring do not genetically inherit the genes encoding the editing components. This phenomenon is known as the dominant maternal effect, as described in Lin and Potter, G3 (2016) doi: 10.1534 / g3.116.034884, the entire contents of which are incorporated herein by reference. In this regard, paternal inheritance of one of the core components (e.g., Cas9 or dgRNA) combined with maternal deposition of compatible components was investigated to determine whether either was sufficient to generate heritable mutations. refer to Figure 1G and 3B , mating between a homozygous Cas9 father and a heterozygous dgRNA-expressing mother was insufficient in F who did not inherit the dgRNA as a gene 1 Mutations (n=12) or knockout phenotypes (N=6, n=252) were induce...

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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 geneticallymodified 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 firstguide 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 firstinsect 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.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to and benefit of U.S. Provisional Application Serial No. 62 / 589,405, filed November 21, 2017, entitled "Novel Sterile Insect Technique," the entire contents of which are incorporated herein by reference. [0003] Statement Regarding Federally Funded Research and Development [0004] This invention was made with government support under Grant Nos. 5K22AI113060 and 1R21AI123937 awarded by the National Institutes of Health and Grant No. HR0011-17-2-0047 awarded by the Defense Advanced Research Projects Agency. The government has certain rights in this invention. Background technique [0005] The mass production and release of sterile males known as the sterile insect technique (SIT) has been used historically to control and eradicate pest populations dating back to the mid-1930s. Previous methods relied on DNA-damaging agents to render them sterile, essentially reducing the overall fitnes...

Claims

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Application Information

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IPC IPC(8): A01K67/033C12N15/09C12N15/63C12N15/85C12N15/90
CPCA01K67/0339C12N9/22C12N15/102A01K2217/058A01K2217/075A01K2217/15A01K2227/706A01K2267/01C12N2310/20C12N15/902C12N2800/80C12N15/8509
Inventor N·P·康杜尔O·S·阿巴里
Owner RGT UNIV OF CALIFORNIA
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