WO2024229156A2 - Procédé de génération de descendance stérile et monosexe - Google Patents

Procédé de génération de descendance stérile et monosexe Download PDF

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Publication number
WO2024229156A2
WO2024229156A2 PCT/US2024/027303 US2024027303W WO2024229156A2 WO 2024229156 A2 WO2024229156 A2 WO 2024229156A2 US 2024027303 W US2024027303 W US 2024027303W WO 2024229156 A2 WO2024229156 A2 WO 2024229156A2
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Prior art keywords
crustacean
mollusk
germ cell
fish
less fish
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PCT/US2024/027303
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WO2024229156A3 (fr
Inventor
Xavier Christophe LAUTH
John Terrell BUCHANAN
Takeshi UMAZUME
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Center for Aquaculture Technologies Inc
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Center for Aquaculture Technologies Inc
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Priority to CN202480045293.7A priority Critical patent/CN121712387A/zh
Priority to KR1020257040229A priority patent/KR20260034684A/ko
Priority to AU2024266019A priority patent/AU2024266019A1/en
Priority to MX2025013109A priority patent/MX2025013109A/es
Priority to EP24800536.5A priority patent/EP4704566A2/fr
Publication of WO2024229156A2 publication Critical patent/WO2024229156A2/fr
Publication of WO2024229156A3 publication Critical patent/WO2024229156A3/fr
Priority to IL324291A priority patent/IL324291A/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/40—Fish
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests

Definitions

  • GE genetically engineered
  • a variety of fish with improved growth rates, food conversion ratios, resistance to disease, and enhanced nutritional benefits, have been developed to address the future demand for seafood and the need to improve sustainability in the aquaculture industry.
  • worldwide adoption of these GE fish is hampered by concerns over their accidental release into natural ecosystems.
  • Cultured fish have been shown to reproduce and survive in natural environments, resulting in feral populations.
  • GE fish may have native relatives, raising the possibility that the genetic modifications will spread throughout the wild population and alter the native gene pool.
  • Commercial GE fish therefore represent a potential threat to the environment and a challenge to policy makers and regulatory agencies tasked with risk- benefit evaluations.
  • triploid fish are produced by applying temperature or pressure shock to fertilized eggs, forcing the incorporation of the second polar body and producing cells with three chromosome sets (3N). Triploid fish do not develop normal gonads Attorney Docket No.133420-285456 IPN: P003PCT as the extra chromosome set disrupts meiosis.
  • IPN IPN
  • triploid induced by physical treatments is triploid induced by genetics, which results from crossing a tetraploid with a diploid fish. Tetraploid fish, however, are difficulty to generate due to poor embryonic survival and slow growth.
  • triploid males produce some normal haploid sperm cells thus allowing males to fertilize eggs, though at a reduced efficiency.
  • negative performance characteristics have been associated with triploid phenotype, including reduced growth and sensitivity to disease.
  • Another approach for sterilizing fish is by hormone treatment extending over several weeks. However, in many cases, including these intensive long-term treatment processes do not have a desirable efficacy of sterility, and/or have been associated with decreased fish growth performance.
  • transgenic-based technologies which include a step of integrating a transgene that induce germ cell death or disrupts their migration patterns resulting in their ablation in developing embryos.
  • transgenes are subject to position effect as well as silencing. Consequently, such approaches are subject to extended regulatory review processes before being considered acceptable for commercial use.
  • Improvements in generating sterile, sex-determined fish, crustaceans, or mollusks is desirable.
  • One or more inventions may reside in a combination or sub-combination of the instrument elements or method steps described below or in other parts of this document.
  • the inventors do not waive or disclaim their rights to any invention or inventions disclosed in this specification merely by not describing such other invention or inventions in the claims.
  • One or more of the previously proposed methods used for sterilizing freshwater and seawater organisms may result in: (1) an insufficient efficacy; (2) increased difficulty to propagate the sterility trait by, for example, having to perform genetic selection to Attorney Docket No.133420-285456 IPN: P003PCT identify a subpopulation of sterile individual, and/or repeating treatment at each generation; (3) an increase in operating costs by, for example, incorporating significant changes in husbandry practices, being untransferable across multiple species, increasing production times, increasing the percentage of sterile organisms with reduced growth and increased sensitivity to disease, increasing mortality rates of sterile organisms, or a combination thereof; (4) gene flow to wild populations and colonization of new habitats by cultured, non- native species; or (4) any combination thereof.
  • the present disclosure provides methods of producing sex-determined sterilized freshwater and seawater organisms by exploiting germline and somatic properties of nucleic acid molecules, for example genes, to produce sterile progeny.
  • One or more examples of the present disclosure may: (1) increase efficacy of sterilization, by for example, allowing mass production of sterile individuals and ensuring that all individuals are completely sterile; (2) decrease operating costs by, for example, decreasing the amount of costly equipment or treatments, being commercially scalable, being transferable across multiple species, decreasing feed, decreasing production times, decreasing the percentage of organisms that attain sexually maturity, increasing the physical size of sexually mature organisms, or a combination thereof; (3) decrease gene flow to wild populations and colonization of new habitats by cultured non-native species; (4) increase culture performance by decreasing loss of energy to gonad development; or (5) any combination thereof, compared to one or more previously proposed methods used for sterilizing freshwater and seawater organisms.
  • the present disclosure also discusses methods of making broodstock freshwater and seawater organisms for use in producing sterilized or sex-determined sterilized freshwater and seawater organisms, as well as the broodstock itself.
  • the present disclosure provides an endogenous germ cell-less fish, crustacean, or mollusk having a gonad that produces gametes with a mutation causing progeny to be sterile.
  • the present disclosure also provides methods of producing said endogenous germ cell-less fish, crustacean, or mollusk, as well as methods of breeding said endogenous germ cell-less fish, crustacean, or mollusk to produce sterile progeny or sterile sex-determined progeny.
  • the present disclosure also provides methods of producing the gonad that produces gametes with a mutation causing progeny to be sterile, as well as the gonad itself.
  • Attorney Docket No.133420-285456 IPN: P003PCT [0014]
  • the present disclosure also provides an endogenous germ cell-less fish, crustacean, or mollusk having a chimeric gonad, where the chimeric gonad comprises at least one transplanted germ cell having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells.
  • the present disclosure also provides methods of producing the gonad that comprises at least one transplanted germ cell having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells, as well as the gonad itself.
  • the present disclosure also provides a method of generating an endogenous germ cell-less fish, crustacean, or mollusk, comprising the steps of: transplanting at least one germ cell having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells, into an endogenous germ cell-less fish, crustacean, or mollusk, producing a chimeric gonad.
  • the present disclosure further provides a method of generating a sterile fish, crustacean, or mollusk, comprising the steps of: breeding (i) an endogenous germ cell-less fish, crustacean, or mollusk as herein disclosed that is female with (ii) an endogenous germ cell-less fish, crustacean, or mollusk as herein disclosed that is male, to produce the sterile fish crustacean, or mollusk.
  • Fig.1 panels A to B is a flowchart showing an example of a herein disclosed method of generating endogenous germ cell-less fish, crustacean, or mollusk having a chimeric gonad and propagating a mutated line.
  • Fig.2 panels A to C is a flowchart showing another example of a herein disclosed method of generating endogenous germ cell-less fish, crustacean, or mollusk having a chimeric gonad and propagating a mutated line.
  • Fig.3 panels A to C is a flowchart showing another example of a herein disclosed method of generating endogenous germ cell-less fish, crustacean, or mollusk having a chimeric gonad and propagating a mutated line.
  • Fig.4 is an illustration showing an example of a herein disclosed germ cell transplantation method to produce functional sperm carrying a miR202 deficient gene (miR- 202-/-). No defects are found during the generation of oogonia and spermatogonia in miR202 null fish progenies obtained from heterozygous miR202mutant parents.
  • miR202 mutant males and females are infertile.
  • the miR202 is expressed in somatic cells surrounding the germ cells (Sertoli and Leydig cells) where it exerts its activity.
  • the lack of miR202 protein causes a defective microenvironment where gamete maturation is impaired.
  • a germline stem cell can be isolated from juvenile miR202 mutant and transplanted into recipient embryos depleted of their own PGCs but carrying a functional miR202 gene.
  • Fig.5 panels A to D are photographs and a graph showing phenotypic characterization of miR202-/- female.
  • Fig.5A shows typical pictures of ovaries in the peritoneal cavity of homozygous miR202 mutant (top part) and WT control (bottom part) tilapia at 7-month of age.
  • Fig.5B shows urogenital papillae from miR202-/- and WT control females.
  • Fig.5C shows a highly magnified light microscopy image of dissected ovary.
  • Fig. 5D shows a graphic representation of the average Gonado Somatic Indexes (GSI) from miR202 mutant female and WT controls.
  • Fig.6 panels A to E are photographs and graphs showing maternal miR202 participate in PGC formation in Nile tilapia.
  • Fig.6B, 6D and 6E illustrate the average number of PGCs in 4-day old embryos ( ⁇ 12 embryos) from mutated females with mosaic (Fig.6B), heterozygous (Fig.6D) and homozygous miR202 mutation in germ cells. There is a significant difference (p ⁇ 0.01) comparing the embryos progeny from wild type control female.
  • FIG.6A and 6C top images represent 4 dpf tilapia embryo progeny of female transgenic line Tg(Zpc5: EGFP: nos 3’UTR) crossed with miR202 mutant males showing a normal PGC count.
  • Bottom images of Fig.6A and 6C represent trunk regions of progenies from Tg(Zpc5: EGFP: nos33’UTR) female lines carrying mosaic miR202 gene mutations or heterozygous miR202 mutations showing reduced PGC count at 4 dpf.
  • the bright dots represent GFP (+) cells (green). The white star points to mislocalized PGCs.
  • Fig.7 panels A to B are illustrations of selected mutation at the miR202 loci.
  • Fig.7A shows the secondary structure tilapia (Oreochromis niloticus) pre miR202 as projected from forna (force-directed RNA) RNA visualization tool (Kerpedjiev, Hammer et al. 2015).
  • Fig.7B shows the location of the miR202 loci in Nile tilapia chromosome LG13.
  • Fig. 7B also show the nucleotide sequence alignment of wild-type and selected mutants with deletions indicated by dashes covering the miR-202-5p region.
  • Fig 8 panels A to C are photographs showing histology of representative of miR202-/- and WT control 7 months ovarian tissue. (Fig.8A and B) representative histology showing stage I oocytes. Bars: 100 micrometers. Fig.8C show histological sections of control ovaries from a WT 7-month-old female. [0027] Fig.9 panels A to C are photographs and graphs showing phenotypic characterization of miR202 -/- male.
  • Fig.9A is typical photographs of dissected testes from homozygous miR202 mutant (miR202 -/- ) and WT tilapia at 7-month of age.
  • Fig.9B is a graph showing the average Gonado Somatic Indexes of miR202 -/- and WT sibling male tilapia at months intervals.
  • Fig.9C is a graph showing the average sperm count from WT and miR202 -/- siblings at 7 months of age. Vertical bars represent standard deviation.
  • Fig.10 panels A to D are photographs showing histological analysis of testes from miR202 mutant (Fig.10C and D) and WT control (Fig.10A and B) at 8 months of age.
  • Fig.10A to C show testicular structure of the whole testis, where miR202 -/- reveal severe depletion of testicular germ cells.
  • Fig.10B is a close-up view of the germinative compartment formed by Sertoli cell surrounding germ cell at different stage of development: SC, spermatocytes; ST, spermatids; and SZ, spermatozoa in the lobule lumen; The germinal compartments are surrounded by steroid-producing Leydig cells (lighter pink coloration).
  • FIG.11 are photographs illustrating histological sections of gonads from Elavl2 ⁇ 8/ ⁇ 8 recipient derived from miR202 mutant germ cells transplantation (GCT) and non- transplanted Elavl2 ⁇ 8/ ⁇ 8 controls. 113 days post transplantation, the recipient fish ovaries and testes were collected and fixed for further histological analysis. Germ cells are absent in non- transplanted control testes and ovaries.
  • Fig.12 panels A to F are photographs illustrating typical morphology and histology of elavl2 -/- tilapia male transplanted with miR202 -/- germ cells (top part) or non- transplanted elavl2 -/- control (bottom panel).
  • Fig.12A morphologically normal testes in the peritoneal cavity of recipient tilapia male
  • Fig.12B translucent testis of non-transplanted male
  • Fig.12C and 12D close-up of box in Fig.12A and 12B under brightfield stereomicroscopy, showing opaque and translucent testes respectively.
  • Fig.12E and 12F show Hematoxylin-eosin-stained histological sections of testes showing that donor derived miR202 -/- germ cell colonized, proliferated, and differentiated in the recipient elavl2 -/- tilapia.
  • Fig.12F shows non-transplanted recipient testes are germ cell free.
  • Fig.13 is photographs showing typical morphology appearance of ovaries from elavl2 -/- tilapia female transplanted with miR202 -/- germ cells (top part) or non- transplanted elavl2 -/- control (bottom panel) at 8 months of age.
  • elavl2 -/- show string like germ cell less ovaries at 7 months of age.
  • Fig.14 panels A and B show illustrations and graphs of crosses between miR202 -/- -GCT female (chimera-gray color) with either WT male (blue color) or miR202 -/- - GCT male (Fig.14A and 14 B top parts).
  • Fig.14A and 14B bottom graphs show the genotypes of their progeny by fin DNA PCR fragment sizing assay utilizing PCR primers that flank the mutation region analysis. The amplification products were sized and detected using capillary electrophoresis.
  • Fig.15 panels A and B are photographs showing the peritoneal cavity of male and female progeny from SSCs miR202- GCT female x miR202-GCT male at time intervals.
  • FIG.16 panels A to C are illustrations showing the genomic region containing Elavl2 and Hermes genes and the selected mutations in these genes.
  • Hermes Rbmps
  • Elavl2 200kb ⁇ 0.2 centimorgan
  • Fig.16A We created a loss of function mutation in both gene
  • Fig.16B Fig.16B is schematics of the tilapia Elavl2 gene
  • Fig.16B is schematics of Hermes gene.
  • Exon (E) are shown as shaded boxes. Arrows point to targeted loci.
  • Figs.16B and 16C show the wild-type reference sequence of ElavI2 (SEQ ID NOs: 116 and 118) and Hermes (SEQ ID NOs: 142 and 144) with the sequence of the selected germ-line mutant alleles: 8 nucleotides deletion for Elavl2 (SEQ ID NOs: 117 and 119) and 16 nucleotides insertion for Hermes (SEQ ID NOs: 143 and 145). Both mutant alleles create truncated proteins that terminate at amino acid 40 and 61 rather than position 372 and 174 respectively.
  • Figs.16B and 16C show the predicted protein sequences of WT and truncated mutant proteins in which the first 12 and 52 amino acids are identical to those of the wild-type Elavl2 and Hermes protein respectively and the following 28 and 9 amino acids are miscoded. Altered amino acids are highlighted.
  • Fig.17 panels A to D are illustrations, graphs, and a photograph showing phenotypic characterization of the progeny from double heterozygous mutant Hermes Ins16/+ , Elavl2 ⁇ 8/+ .
  • Fig.17A shows the expected mendelian distribution of genotypes from completely linked genes (Elavl2 and Hermes) in which h represents a lethal receive Hermes mutation.
  • Fig.17B graphically shows the percentage of developing embryos 0-3 days post fertilization (blue columns represent the mean and vertical bar the standard deviation) and percentage of deformed embryos ( ⁇ 20%, yellow column).
  • Fig.17C is a photograph of 9dpf hatchlings showing Hermes Ins16/Ins16 escapes (EEhh) having cranio-facial and body axis deformities and WT control (right image).
  • FIG.18 are photographs showing hatchlings progeny with normal pigmentation or amelanic typical of the homozygous tyr-/- albino mutation. Offspring from normally pigmented surrogate parents transplanted with donor germ cells from Tyr-/- mutant consistently exhibit albino pigmentation.
  • Panel A is photographs showing dissected ovaries from 9-month-old sibling female tilapia, distinguishing between those heterozygous for the miR202 gene (fertile, with ripe ovaries) and those homozygous mutant (sterile, with ovaries arrested at the previtellogenic stage).
  • the present disclosure provides an endogenous germ cell-less fish, crustacean, or mollusk having a chimeric gonad, wherein the chimeric gonad comprises at least one transplanted germ cell having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells.
  • the present disclosure also provides a method of generating an endogenous germ cell-less fish, crustacean, or mollusk, comprising the steps of: transplanting at least one germ cell having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells, into an endogenous germ cell-less fish, crustacean, or mollusk, producing a chimeric gonad.
  • the present disclosure also provides a method of generating a sterile sex-determined fish, crustacean, or mollusk.
  • the method comprises the step of: breeding (i) a fertile female fish, crustacean, or mollusk having a homozygous mutation with (ii) a fertile male fish, crustacean, or mollusk having a homozygous mutation to produce the sterile sex-determined fish, crustacean, or mollusk.
  • the mutation directly or indirectly disrupts spermiogenesis, and/or that directly disrupts vitellogenesis.
  • the fertility of the fertile female fish, crustacean, or mollusk and the fertile male fish, crustacean, or mollusk have been rescued.
  • the present disclosure also provides a method of generating a sterile fish, crustacean, or mollusk, comprising the steps of: breeding (i) an endogenous germ cell-less fish, crustacean, or mollusk as herein disclosed that is female with (ii) an endogenous germ cell-less fish, crustacean, or mollusk as herein disclosed that is male, to produce the sterile fish crustacean, or mollusk.
  • the present disclosure also provides a method of generating a sterile fish, crustacean, or mollusk, comprising the steps of: breeding (i) an endogenous germ cell-less fish, crustacean, or mollusk produced by the herein disclosed methods that is female with (ii) an endogenous germ cell-less fish, crustacean, or mollusk produced by the herein disclosed method that is male, to produce the sterile fish crustacean, or mollusk.
  • the present disclose also provides an endogenous germ cell-less fish, crustacean, or mollusk having a chimeric gonad, wherein the chimeric gonad comprises at least one transplanted: a) Oogonial stem cell (OSC) from a homogametic female donor, such as XX, having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells; or b) Spermatogonial stem cell (SCC) from a homogametic male donor, such as ZZ, having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells.
  • OSC Oogonial stem cell
  • SCC Spermatogonial stem cell
  • the present disclosure also provides a method of generating an endogenous germ cell-less fish, crustacean, or mollusk, comprising the steps of: transplanting at least one: a) Oogonial stem cell (OSC) from a homogametic female donor, such as XX, having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells; or b) Spermatogonial stem cell (SCC) from a homogametic male donor, such as ZZ, having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells, into an endogenous germ cell-less fish, crustacean, or mollusk, producing a chimeric gonad.
  • OSC Oogonial stem cell
  • SCC Spermatogonial stem cell
  • the present disclosure also provides an endogenous germ cell-less fish, crustacean, or mollusk having a chimeric gonad, wherein the chimeric gonad comprises at least one transplanted: a) Oogonial stem cell (OSC) from a homogametic superfemale donor, such as WW, having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells; or b) Spermatogonial stem cell (SCC) from a homogametic supermale donor, such as YY, having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells.
  • OSC Oogonial stem cell
  • SCC Spermatogonial stem cell
  • the present disclosure also provides a method of generating an endogenous germ cell-less fish, crustacean, or mollusk, comprising the steps of: transplanting at least one: a) Oogonial stem cell (OSC) from a homogametic superfemale donor, such as WW, having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells; or b) Spermatogonial stem cell (SCC) from a homogametic supermale donor, such as YY, having a mutation that is present in the germline and disrupts Attorney Docket No.133420-285456 IPN: P003PCT the development and/or function of somatic gonadal cells, into an endogenous germ cell-less fish, crustacean, or mollusk, producing a chimeric gonad.
  • OSC Oogonial stem cell
  • SCC Spermatogonial stem cell
  • the superfemale such as WW
  • OSC Oogonial stem cell
  • the supermale such as YY
  • YY is generated by: transplanting at least one Spermatogonial stem cell (SCC) from a heterogametic male donor, such as XY, having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells, into a male and a female endogenous germ cell-less fish, crustacean, or mollusk, producing a chimeric gonad; breeding the male endogenous germ cell-less fish, crustacean, or mollusk having a chimeric gonad with the female endogenous germ cell-less fish, crustacean, or mollusk having a chimeric gonad; and selecting a progeny that is homogametic by genotypic selection.
  • SCC Spermatogonial stem cell
  • the present disclosure also provides a method of generating a sterile sex- determined fish, crustacean, or mollusk, comprising the steps of: breeding (i) an endogenous germ cell-less fish, crustacean, or mollusk as herein disclosed that is female with (ii) an endogenous germ cell-less fish, crustacean, or mollusk as herein disclosed that is male and from the same sex-determination system as the (i) female, to produce the sterile sex- determined fish, crustacean, or mollusk.
  • the present disclosure also provides a method of generating a sterile sex- determined fish, crustacean, or mollusk, comprising the steps of: breeding (i) an endogenous germ cell-less fish, crustacean, or mollusk produced by the herein disclosed methods that is female with (ii) an endogenous germ cell-less fish, crustacean, or mollusk produced by the herein disclosed methods that is male and from the same sex-determination system as the (i) female, to produce the sterile sex-determined fish, crustacean, or mollusk.
  • a fish refers to any gill-bearing craniate animal that lacks limbs with digits.
  • a crustacean refers to any arthropod Attorney Docket No.133420-285456 IPN: P003PCT taxon. Examples of crustaceans are crabs, lobsters, crayfish, and shrimp.
  • a mollusk refers to any invertebrate animal with a soft unsegmented body usually enclosed in a calcareous shell. Examples of mollusks are clams, scallops, oysters, octopus, squid and chitons.
  • the fish, crustacean, or mollusk may be an Atlantic salmon, Rainbow Trout, Coho Salmon, tilapia, cobia, Seriola spp., Grouper, Snapper, barramundi, Sea Bream, Sea Bass, lumpfish, sturgeon, Litopeneus vannamei, Peneaus monadon, oysters, clams or mussels.
  • a sterile fish, crustacean, or mollusk refers to any fish, crustacean, or mollusk with a diminished ability to generate progeny through breeding or crossing or mixing gametes as compared to its wild-type counterpart; for example, a sterile fish, crustacean, or mollusk may have an about 50%, about 75%, about 90%, about 95%, or 100% reduced likelihood of producing viable progeny.
  • a fertile fish, crustacean, or mollusk refers to any fish, crustacean, or mollusk that possesses the ability to produce progeny through breeding or crossing or mixing gametes.
  • breeding and crossing refers to any process in which a male of a species and a female of a species mate or their gametes are mixed to produce fertile eggs, progeny or offspring.
  • a sex-determined fish, crustacean, or mollusk refers to any fish, crustacean, or mollusk progeny in which the sex of the progeny has been pre-determined by disrupting the progeny’s sexual differentiation pathway. In some examples, sex-determined progeny of the same generation are monosex.
  • Chimeric gonad refers to a gonad that has been colonized with one or more germ cells from a genotype distinct from the genotype housing the gonad.
  • Germ cells refers to any biological cell that gives rise to the gametes of an organism.
  • the germline refers to a population germ cells that pass on their genetic material to the progeny.
  • Somatic cells refers to any cell that is not a gamete, germ cell, or stem cell, and therefore does not pass through generations.
  • Somatic gonadal cells refer to somatic cells that generate the various cell types within the testis or ovary that support gametogenesis.
  • a mutation present in the germline and disrupts the development and/or function of somatic gonadal cells refers to any genetic mutation of a nucleic acid molecule that is passed from one generation to another and that directly or indirectly somatically modulates gonadal development and/or function.
  • Directly or indirectly modulating refers to: (1) mutating the coding sequence of one or more nucleic acid molecules that is passed from one generation to another and that directly or indirectly somatically modulates gonadal Attorney Docket No.133420-285456 IPN: P003PCT development and/or function; (2) mutating a non-coding sequence that has at least some control over the transcription of one or more nucleic acid molecules that is passed from one generation to another and that directly or indirectly somatically modulates gonadal development and/or function; (3) mutating the coding sequence of another gene or nucleic acid molecule that is involved in post-transcriptional regulation of one or more nucleic acid molecules that is passed from one generation to another and that directly or indirectly somatically modulates gonadal development and/or function; or (4) any combination thereof.
  • a mutation may be any type of alteration of a nucleotide sequence of interest, for example, nucleotide insertions, nucleotide deletions, and nucleotide substitutions.
  • the mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells may be a mutation in microRNA miR202 or an ortholog thereof.
  • Somatically modulating gonadal development and/or function refers to disrupting the maturation of gonadal somatic cells and/or disrupting the interaction between gonadal somatic cells and germ cells in the gonad.
  • Examples include disrupting gametogenesis; disrupting the development and/or function of testis and/or ovary cells; disrupting the development and/or function of Sertoli, Theca, Granulosa, and/or Leydig cells; disrupting at least one signaling molecule produced by Sertoli, Theca, Granulosa, and/or Leydig cells; disrupting at least one secreted diffusible signal protein or growth factor protein that regulates Spermatogonial stem cell (SCC) and/or Oogonial stem cell (OSC) renewal and/or differentiation.
  • SCC Spermatogonial stem cell
  • OSC Oogonial stem cell
  • the at least one secreted diffusible signal protein or growth factor protein may be glial cell line-derived neurotrophic factor (GDNF); bone morphogenetic protein 4 (BMP4); stem cell factor (SCF); fibroblast growth factor 2 (FGF2); C-X-C motif chemokine 12 (CXCL12); and/or epidermal growth factor (EGF).
  • GDNF glial cell line-derived neurotrophic factor
  • BMP4 bone morphogenetic protein 4
  • SCF stem cell factor
  • FGF2 fibroblast growth factor 2
  • CXCL12 C-X-C motif chemokine 12
  • EGF epidermal growth factor
  • the germ stem cell transplantation is a process comprising: obtaining one or more germline stem cells, for example from about 1 to about 6,000 germ cells, or from about 500 to about 6,000 germ cells, from a fertile homozygous male or female fish, crustacean, or mollusk having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells; and transplanting the one or more germline stem cells into an endogenous germ cell-less recipient male or female fish, crustacean, or mollusk.
  • one or more germline stem cells for example from about 1 to about 6,000 germ cells, or from about 500 to about 6,000 germ cells, from a fertile homozygous male or female fish, crustacean, or mollusk having a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells.
  • a recipient male or female fish, crustacean, or mollusk is any embryo depleted of their own germ cells Attorney Docket No.133420-285456 IPN: P003PCT but homozygous wild type in the germline at the locus for the mutation that is present in the donor male or female fish, crustacean, or mollusk.
  • the donor male or female fish, crustacean, or mollusk can be a juvenile or adult fish.
  • a juvenile fish, crustacean, or mollusk is a stage of maturation that is prior to development and/or production of hormones related to sexual maturation or puberty; is at an age of from about 1 month to about 9 months, for example, from about 6 months to about 9 months; and/or has a gonadosomatic index (GSI) of from about 0.1% to about 0.5%.
  • the recipient after transplantation is a fish, crustacean or mollusk having a chimeric gonad with normal somatic cells but a mutant germline.
  • the at least one germ cell is transplanted into the peritoneal cavity of the embryo or hatchling of the recipient, for example, the endogenous germ cell-less fish, crustacean, or mollusk.
  • a germ cell-less recipient may be created using genetic mutation, ploidy manipulation, such as triploidy manipulation, hybridization strategies, exposure to high levels of sex hormones, using morpholinos to disrupt primordial germ cell development, using sterile hybrids, exposure to chemicals and high temperature. See examples described in: Hunter et al, 1982; Solar et al, 1984; Piferrer et al, 1994; Hunter, G.A., E.M. Donaldson, F.W. Goetz, and P.R. Edgell.1982. Production of all-female and sterile Coho salmon, and experimental evidence for male heterogamety. Transactions of the American Fisheries Society 111: 367-372; Piferrer, F, M Carillo, S.
  • Germ Cell-Less Hybrid Fish Ideal Recipient for Attorney Docket No.133420-285456 IPN: P003PCT Spermatogonial Transplantation for the Rapid Production of Donor-Derived Sperm. Biol. Reprod.2019, 101, 492–500.
  • Triploidization see examples described in: Lee, S.; Bang, W.Y.; Yang, H.S.; Lee, D.S.; Song, H.Y. Production of Juvenile Masu Salmon (Oncorhynchus masou) from Spermatogonia-Derivedsperm and Oogonia-Derived Eggs via Intraperitoneal Transplantation of Immature Germ Cells. Biochem. Biophys. Res.
  • the at least one germ cell may be a Spermatogonial stem cell (SCC); an Oogonial stem cell (OSC).
  • SCC Spermatogonial stem cell
  • OSC Oogonial stem cell
  • the at least one germ cell may be from a heterogametic male donor, such as XY; from a homogametic female donor, such as XX; from a homogametic male donor, such as ZZ; from a heterogametic female donor, such as WZ; from a homogametic superfemale donor, such as WW; or from a homogametic supermale donor, such as YY.
  • At least one herein disclosed Oogonial stem cell (OSC) from a female donor is transplanted into a herein disclosed endogenous germ cell-less fish, crustacean, or mollusk that is female, and into a herein disclosed endogenous germ cell-less fish, crustaceans, or mollusk that is male; and the male and female recipient fish, crustacean or mollusks are bred producing sterile fish, crustaceans, or mollusks.
  • OSC Oogonial stem cell
  • At least one herein disclosed Spermatogonial stem cell (SCC) from a male donor is transplanted into a herein disclosed endogenous germ cell-less fish, crustacean, or mollusk that is female, and into a herein disclosed endogenous germ cell-less fish, crustaceans, or mollusk that is male; and the male and female recipient fish, crustacean or mollusks are bred producing sterile fish, crustaceans, or mollusks.
  • SCC Spermatogonial stem cell
  • At least one herein disclosed Oogonial stem cell (OSC) from a homogametic female donor, such as XX, is transplanted into a herein disclosed endogenous germ cell-less fish, crustacean, or mollusk that is female, and into a herein disclosed endogenous germ cell-less fish, crustaceans, or mollusk that is male; and the male and female recipient fish, crustacean or mollusks are bred producing sterile fish, crustaceans, or mollusks that are female.
  • OSC Oogonial stem cell
  • the superfemale such as WW
  • OSC Oogonial stem cell
  • At least one herein disclosed Spermatogonial stem cell (SCC) from a homogametic male donor, such as ZZ, is transplanted into a herein disclosed endogenous germ cell-less fish, crustacean, or mollusk that is female, and into a herein disclosed endogenous germ cell-less fish, crustacean, or mollusk that is male; and the male and female recipient fish, crustacean or mollusks are bred producing sterile fish, crustaceans, or mollusks that are male.
  • SCC Spermatogonial stem cell
  • At least one herein disclosed Spermatogonial stem cell (SCC) from a homogametic supermale donor, such as YY, is transplanted into a herein disclosed endogenous germ cell-less fish, crustacean, or mollusk that is female, and into a herein disclosed endogenous germ cell-less fish, crustacean, or mollusk that is male; and the male and female recipient fish, crustaceans or mollusks are bred producing sterile fish, crustaceans, or mollusks that are male.
  • SCC Spermatogonial stem cell
  • the supermale such as YY
  • SCC Spermatogonial stem cell
  • Fig.1 is a flowchart showing an example of a herein disclosed method of generating endogenous germ cell-less fish, crustacean, or mollusk having a chimeric gonad and propagating a mutated line, i.e., generating a sterile fish, crustacean, or mollusk therefrom.
  • the mutant donor has a mutation that is present in the germline and disrupts the Attorney Docket No.133420-285456 IPN: P003PCT development and/or function of somatic gonadal cells. In this example, the mutation is in miR202.
  • the mutation causes a defect in the microenvironment or niche of the testis and ovary resulting in germ cell development arrest and infertility in adult homozygous mutant males and females (Fig.1A).
  • a chimera using germ cell transplantation is produced.
  • ovarian or testicular cell suspensions obtained from a homozygous mutant fish, crustacean, or mollusk is transplanted into the peritoneal cavity of an endogenous germ cell-less recipient embryo or hatchling of a fish, crustacean, or mollusk that are wild type for the mutation producing a chimeric gonad.
  • the host chimeric recipient has normal somatic cells and a mutant germ line. In the chimeric recipients, the mutation is silent and does not block germ cell development.
  • the chimeric recipients have ovaries and testes capable of nurturing mutant germ cells and only produce donor derived gametes carrying the mutated genes (Fig.1B).
  • the recipient fish, crustacean, or mollusk can be used as commercial broodstock for mass production of sterile fish, crustaceans or mollusks.
  • Fig.2 is a flowchart showing an example of a herein disclosed method of generating endogenous germ cell-less fish, crustacean, or mollusk having a chimeric gonad and propagating a mutated line, i.e., generating monosex sterile fish, crustacean, or mollusk.
  • the mutant donor has a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells. In this example, the mutation is in miR202.
  • XY Spermatogonial Stem Cells
  • OSCs Oogonial Stem Cells
  • Incrossing recipients will produce offspring having different male-to-female sex ratio: following SSC transplantation, Y eggs will be produced, leading to XX, XY and YY at a mendelian distribution of 1:2:1 ratio; and following OSCs (XX) transplantation, male and female producing gametes containing X chromosome only will be produced. Incrossing germ cell transplanted recipients derived from OSCs will result exclusively in female offspring production (Fig.2B). When SSCs (YY) are transplanted, only Y gametes will be produced resulting in sperm and eggs containing Y chromosomes. Incrossing recipients from this group will produce 100% male infertile progeny (Fig.2C).
  • Fig.3 is a flowchart showing an example of a herein disclosed method of generating endogenous germ cell-less fish, crustacean, or mollusk having a chimeric gonad and propagating a mutated line, i.e., generating monosex sterile fish, crustacean, or mollusk.
  • the mutant donor has a mutation that is present in the germline and disrupts the development and/or function of somatic gonadal cells. In this example, the mutation is in miR202.
  • ZZ Spermatogonial Stem Cells
  • OSCs Oogonial Stem Cells
  • Incrossing germ cell transplanted recipients will produce offspring having different male-to-female sex ratio: following OSC transplantation, W sperm will be produced, leading to ZZ, ZW and WW at a mendelian distribution of 1:2:1 ratio, resulting in 75% female progeny and following SSCs (ZZ) transplantation, male and female producing gametes containing Z chromosome only will be produced.
  • Incrossing ZZ germ cell transplanted recipients derived from SSCs will result exclusively in male offspring production (Fig.3A).
  • OSCs from superfemale (WW) are transplanted, only W gametes will be produced, resulting in sperm and eggs containing W chromosomes.
  • Incrossing recipients from this group will produce female only progeny (Fig.3C).
  • the herein disclosed propagating and incrossing may be performed by mixing sperm and eggs.
  • the present inventors surprisingly found that miR202 mutant tilapia lines developed penetrant male and female infertility phenotypes. Considering that the same gene mutation did not impair zebrafish fertility and only caused partial infertility in medaka, the high level of infertility described here was unexpected.
  • the present inventors further discovered that despite the existence of mRNA targets for miR202 regulation within germ cells, infertility in tilapia resulted from the loss of miR202 function in somatic gonadal cells.
  • Tilapia (Oreochromis niloticus) lines used in this study are derived from a Brazilian strain and carry a transgene where an oocyte specific promoter was functionally linked to a “eGFP:nos33’UTR” cassette.
  • the resulting transgenic female line express the Green Fluorescent Protein (GFP) in the PGCs of their offspring embryos.
  • GFP Green Fluorescent Protein
  • Tilapia were housed in multiple recirculating aquatic holding systems to accommodate all life stages. The culture system was maintained at 27oC (12H light: 12H dark).
  • Gene Editing The miR202 KO tilapia were generated with genome editing technology.
  • the tilapia miR202 gene (MiRBase; Ensemble: ENSONIG00000021943) is located in an intergenic region on the Linkage group 13 (putative chromosome).
  • the flanking sequence 5’-GTATGTGCATAGGAAAA-3’ was selected as a single guide RNA to target the tilapia miR-202-5p seed sequence.
  • These founders were genotyped by PCR fragment analysis. Tilapia lines carrying a 7, 8 and 19-bp deletions (TTCCTTT and TTTTCCTA and TCCTTTTTCCTATGCACAT) around the miR202 seed sequence were selected and bred for this project (see Fig.7).
  • Fluorescence PCR for F0 genotyping PCR reactions used 3.8 ⁇ L of water, 0.2 ⁇ L of fin-DNA and 5 ⁇ L of PCR master mix (Quiagen Multiplex PCR) with 1 ul of primer mix consisting of the following three primers: the Labeled tail primer with fluorescent tag (6- FAM, NED), amplicon-specific forward primer with forward tail (5′ - TGTAAAACGACGGCCAGT-3′ and 5′ -TAGGAGTGCAGCAAGCAT-3′) amplicon-specific reverse primer (5’-GTTCCAGTGTCCAGAATCGGG-3’ and 5’-CTGGTGGAATACCTCTGC- 3’).
  • the Labeled tail primer with fluorescent tag (6- FAM, NED
  • amplicon-specific forward primer with forward tail (5′ - TGTAAAACGACGGCCAGT-3′ and 5′ -TAGGAGTGCAGCAAGCAT-3′
  • amplicon-specific reverse primer 5’-GTTCCAGTGTCCAGAATCGGG-3’
  • PCR conditions were as follows: denaturation at 95°C for 15 min, followed by 30 cycles of amplification (94°C for 30 sec, 57°C for 45 sec, and 72°C for 45 sec), followed by 8 cycles of amplification (94°C for 30 sec, 53°C for 45 sec, and 72°C for 45 sec) and final extension at 72°C for 10 min, and an indefinite hold at 4°C.
  • One-two microliters of 1:10 dilution of the resulting amplicon were resolved via capillary electrophoresis (CE) with an added LIZ labeled size standard to determine the amplicon sizes accurate to base-pair resolution (Retrogen Inc., San Diego).
  • CE capillary electrophoresis
  • LIZ labeled size standard to determine the amplicon sizes accurate to base-pair resolution
  • the size of the peak relative to the wild-type peak control determines the nature (insertion or deletion) and length of the mutation.
  • the number of peak(s) indicate the level of mosaicism.
  • We selected F0 mosaic founder carrying the fewest number of mutant Attorney Docket No.133420-285456 IPN: P003PCT alleles (2-4 peak preferentially).
  • the allele sizes were used to calculate the observed indel mutations. Mutations that are not in multiples of 3 bp and thus predicted to be frameshift mutations were selected for further confirmation by sequencing except for mutation in the non-coding sequence of genes targeted. Mutations of size greater than 8bp but smaller than 30bp were preferentially selected to ease genotyping by QPCR melt analysis for subsequent generations.
  • sequence confirmation the PCR product of the selected indel is further submitted to sequencing. Sequencing chromatography of PCR showing two simultaneous reads are indicative of the presence of indels. The start of the deletion or insertion typically begins when the sequence read becomes divergent. The dual sequences are then carefully analyze to detect unique nucleotide reads. The pattern of unique nucleotide read is then analyzed against series of artificial single read patterns generated from shifting the wild type sequence over itself incrementally.
  • the tilapia Zpc5 promoter is an oocyte-specific promoter, active during oogenesis prior to the first meiotic division.
  • all embryos from a heterozygous or homozygous transgenic female inherit the eGFP:tnos 3’UTR mRNA, which localizes and becomes expressed exclusively in PGCs through the action of cis-acting RNA elements in their 3’UTR (tilapia nos33’UTR).
  • Embryos (4 days post fertilization) were euthanized by an overdose of tricaine methanesulfonate (MS-222, 200-300mg/l) by prolonged immersion for at least 10 minutes.
  • Stock preparation is 4g/L 10 buffered to pH 7 in sodium bicarbonate (at 2:1 bicarb to MS-222).
  • the embryos were transferred onto a glass surface in PBS and their yolk removed. Deyolked embryos were squashed between a microscope slide and a cover slip and analyzed under fluorescent microscopy equipped with camera for imaging. Images were taken using either bright field or epifluorescent light with an enhanced green fluorescent protein filter.
  • F1 genotyping The selected male founders were crossed with tilapia female carrying the ZPC5:eGFP:tnos 3’UTR construct. Their F1 progeny were raised to 2 months of age, anesthetized by immersion in 200mg/L MS-222 (tricaine) and transferred onto a clean surface using a plastic spoon. Their fin was clipped with a razor blade, and placed onto a well (96 well plate with caps). Fin clipped fish were then placed in individual jars while their fin DNA was analyzed by fluorescence PCR. In brief, 60 ⁇ l of a solution containing 9.4% Chelex and 0.625mg/ml proteinase K is added to each well for overnight tissue digestion and gDNA extraction in a 55°C incubator.
  • gDNA Attorney Docket No.133420-285456 IPN: P003PCT extraction solution was then diluted 10 ⁇ with ultra-clean water to remove any PCR inhibitors in the mixture.
  • the herein disclosed crossing may be performed by mixing sperm and eggs.
  • the qPCR was performed using 40 cycles of 15 seconds at 95°C, 60 seconds at 60°C, followed by melting curve analysis to confirm the specificity of the assay (67°C to 97°C).
  • short PCR amplicons (approximately 120–200 bp) that include the region of interest are generated from a gDNA sample, subjected to temperature-dependent dissociation (melting curve).
  • melting curve temperature-dependent dissociation
  • Sperm were counted using a Neubauer hemocytometer slide, as well as by spectrophotometry (optical density (O.D) at 600nm) of serially diluted samples.
  • Sperm motility was measured in terms of percent motile spermatozoa in field of view [1].
  • Morphology of the sperm cells stained with eosin-nigrosin was analyzed under light microscopy at 400x. Fertilization capacity of sperm was assayed by in vitro fertilization of wild type eggs from 3 different females at the optimal sperm to egg ratio (100 eggs for 5.106 spermatozoa). Wild type egg quality was tested in parallel using sperm from WT males.
  • Fertilization rates were expressed as a percentage of Attorney Docket No.133420-285456 IPN: P003PCT surviving embryos to total eggs collected at 24hrs post fertilization. The mean values obtained from these studies were compared across mutant genotypes using an unpaired t- test.
  • Assessment of sterility in females We recorded the body weight of all fish sampled. A minimum of six females for each genotype was dissected at 4 and 6 months of age and their gonads photographed in situ before dissection. The mean total gonadosomatic index was statistically compared across all genotypes (unpaired T-test).
  • Donor cell isolation and germ cell transplantation Germ cell stem cells were harvested from the gonads of 3-4 months old fish ( ⁇ 50-70g) through enzymatic digestion as described by Lacerda [3]. In brief, the freshly isolated gonads were minced and incubated in 1 ml of 0.5 % trypsin (Worthington Biochemical Corp., Lakewood, NJ) in PBS (pH 8.2) containing 5 % fetal bovine serum (Gibco Invitrogen Co., Grand Island, NY) and 0.05 % DNase I (Roche Diagnostics, Mannheim, Germany) for 3-4 h at 25 °C.
  • Hapas spawning experiments In each hapa, three wildtype (WT) females 9- 12 months of age with an average body weight of 700g were paired with one male chosen from two genotypes (miR202+/- and miR202-/-), all originating from heterozygous parents and possessing an average body weight of 800g at 12mpf (mpf: months post fertilization). The fish were permitted to engage in natural spawning for a maximum period of 30 days. Monitoring of female fish occurred every other day to detect the presence of eggs in the buccal cavity. Upon discovery, females carrying eggs were captured, and the eggs were extracted from their mouths.
  • the pit tag number of each egg-bearing female was recorded, Attorney Docket No.133420-285456 IPN: P003PCT and they were subsequently replaced with new WT females.
  • the total number of eggs from each spawn was tallied, and the fertile embryos were then incubated in petri dishes until reaching stages 14–16 during the pharyngula period. For each spawn, the ratio of the total number of eggs to live embryos at the pharyngula stage was recorded.
  • Germ cell-free recipient larvae (5-7dpf) were anesthetized with 0.01 % ethyl 3-aminobenzoate methanesulfonate salt (Sigma-Aldrich Inc.) and transferred to a Petri dish coated with 2 % agar.
  • Transplantation needles were prepared by pulling glass capillaries using an electric puller (PB-7, Narishige). The tips of the needles were sharpened with a grinder (EG-4, Narishige) until the opening reached 30 ⁇ m.
  • Example 2 Generation of founder lines and associated phenotypes.
  • miR202 engineered nucleases to abrogate its function. We targeted a site in precursor miR202 gene adjacent to the seed sequence. Alongside the miR202 sequence, we co-target a pigmentation gene (Tyrosinase: tyr) to serve as a mutagenesis selection marker.
  • tyr pigmentation gene
  • miR202+/- mutant mothers produce a progeny with a 50% reduction in PGC count irrespective of the embryo genotypes and that of the father (Fig.6D). This result confirmed that this gene dosage sensitivity is maternal specific with no paternal or zygotic effect.
  • miR202 is maternally Attorney Docket No.133420-285456 IPN: P003PCT expressed in oocyte, stored as a maternal transcript in unfertilized eggs and act as a critical constituent of the germ plasm in developing embryos.
  • Example 4 - Tilapia miR202 is required for female reproductive success.
  • F1 fish carrying the 7-bp, 8-bp and 15-bp deletions were used to generate stable heterozygous F2 line for each allele. Incrossing F2s produced homozygous for each mutation at the expected Mendelian ratio (25%). We observed no external morphological anomalies in any genotype produced except for underdeveloped urogenital papillae in miR202-/- homozygotes (Fig.5B). We dissected controls (miR202+/+ and miR202+/ ⁇ ) and homozygous mutant females at time intervals and analyzed the gross morphology and histology of their gonads (Fig.5).
  • the herein disclosed incrossing may be performed by mixing sperm and eggs.
  • Example 5 - Tilapia Mir202 is required for male reproductive success.
  • homozygous mutation of miR202 gene led to the development of atrophic male urogenital papillae, as well as smaller and translucent testis (Fig.9A).
  • the miR202-/- males were removed from the breeding tank and replaced with fertile miR202+/- sibling control males from the same parental cross (miR202+/- parents). The number of spawns and fertilization success rates were record. In some examples, the herein disclosed crossing may be performed by mixing sperm and eggs. [0095] Altogether our result indicates highly penetrant male spermatogenic disruption associated with male subfertility. [0096] Example 6 - Tilapia reproductive capacity is controlled by miR202 expression in somatic gonadal cells. [0097] Our results showing that miR202 is a germ plasm-specific RNA involved in PGC survival and migration, indicate that miR202 is maternally expressed in tilapia oocytes.
  • miR202 play a key regulatory role at different developmental stages of the male and female gonads. Whether the gonadal somatic or the germ cells miR202 expression are causative in determining infertility remains unclear.
  • To analyze the tissue specific contribution of miR202 to germ cell production we created a chimera made of miR202+/+ somatic cell and miR202-/- germ cells. Our approach to generate germ-line replacement chimeras is outlined in Fig.4.
  • dnd1 or Elavl2 heterozygous progeny of dnd1 or Elavl2 heterozygous, and only raised transplanted dnd1-/- [5] or Elavl2-/- [5] and non-transplanted controls to adulthood.
  • the dnd1 and Elavl2 alleles are a fully penetrant recessive zygotic sterile mutation, causing loss of endogenous germ cells (Figs.11-13). In these mutants, the gonadal microenvironment is believed to be normal as the genes are exclusively expressed in germ cells.
  • Example 7 - Tilapia reproductive capacity is controlled by miR202 expression in somatic gonadal cells.
  • GCT germ cell transplanted
  • Example 8 - miR202-GCT broodstock can mass produce infertile progeny.
  • the progeny of GCT-female x GCT-male consisted of embryos with the completely white phenotype typical of the homozygous tyr-/- albino mutation (Fig.18). This demonstrates that germ cells from recipient had the Tyr mutant alleles, because completely albino could only result from matings in which both male and female gametes had the Tyr mutation.
  • To further confirm the genotypes of the progeny we analyzed DNA from 100 embryos progeny resulting from the mating of a female and a male recipient. Of the 3x30 progenies from such intercrosses and incrosses, 3x30 heterozygote (miR202+/-) and 3x30 homozygous miR202-/- mutant were identified.
  • Table 3 displays the sex ratio resulting from germ cell-transplanted male and female tilapia that received spermatogonia (XY), as well as control crosses between GCT males and WT (XX) females. Phenotypic sex was determined at 3 months of age through morphological assessment of the urogenital papillae and dissection of the gonads. In some examples, the herein disclosed crossing or incrossing may be performed by mixing sperm and eggs.
  • Example 9 - Surrogate broodstock can generate progeny populations consisting predominantly of either males or females, depending on the sex of the donor.
  • All batches of offspring exhibited normal development, differentiating into phenotypic males and females, with sex ratios strongly biased towards either males (>73%) or females (>95%) (see Table 3 and Table 4, respectively). This bias depended on whether the transplanted germ cells were oogonia or spermatogonia. Such biased sex ratios were anticipated since the germ-cell grafts were created using testicular extract (XY germ cells) or ovarian extract (XX germ cells), transplanted into sterile male and female recipient hatchlings.
  • progeny resulting from oogonia-injected recipients possessed only X chromosomes and all developed into XX females, with frequencies ranging from 92 to 100% (see Table 4).
  • Table 4 female tilapia that received oogonia (XY), as well as control crosses between GCT female and WT (XY) males. Phenotypic sex was determined at 3 months of age through morphological assessment of the urogenital papillae and dissection of the gonads. Attorney Docket No.133420-285456 IPN: P003PCT [00112] Table 5: [00113] Table 5 presents the fertilization rate, as well as the percentage of males and females obtained from potential YY males of Nile tilapia (derived from a cross as described in Table Y1) paired with a WT female.
  • Phenotypic sex was determined at 3 months of age through morphological assessment of the urogenital papillae and dissection of the gonads. In some examples, the herein disclosed crossing may be performed by mixing sperm and eggs.
  • Example 10 Sterile female (miR202-/-) fish surpassed fertile controls (miR202+/-) in performance.
  • SEQ ID NO: 162 (MIR202 WILD-TYPE ALLELE) LENGTH: 94bp TYPE: genomic DNA (SEQ ID NO:162)
  • ORGANISM Nile tilapia 1 CTCGCTGTTCCTTTTTCCTATGCACATACTTCTTTGAGATTTAACTTTAAAGAGGCATAA 60 briefly............................ GGCATGGGAAAATGGGGCTGCAGAGGTATTCCAC 94 ..2015.
  • SEQ ID NO: 163 (MIR202 MUTANT ALLELE - 7nt deletion) LENGTH: 87bp (-7pb)
  • TYPE genomic DNA (SEQ ID NO: 163)
  • ORGANISM Nile tilapia 1 CTCGCTGTTCCTATGCACATACTTCTTTGAGATTTAACTTTAAAGAGGCATAAGGCATGG 60 .............................................
  • SEQ ID NO: 164 (MIR202 MUTANT ALLELE- 8nt deletion) LENGTH: 86bp (-8pb) TYPE: genomic DNA (SEQ ID NO: 164)
  • ORGANISM Nile tilapia 1 CTCGCTGTTCCTTGCACATACTTCTTTGAGATTTAACTTTAAAGAGGCATAAGGCATGGG 60 ............................................. AAAATGGGGCTGCAGAGGTATTCCAC 86 ..........................

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Abstract

L'invention concerne un poisson, un crustacé ou un mollusque sans cellule germinale endogène ayant une gonade chimérique, la gonade chimérique comprenant au moins une cellule germinale transplantée ayant une mutation qui est présente dans la lignée germinale et interrompt le développement et/ou la fonction de cellules gonadiques somatiques, qui peuvent être utilisés en tant que géniteurs. L'invention concerne également des procédés pour la production de poissons d'élevage, de crustacés ou de mollusques destinés à être utilisés pour produire des poissons, des crustacés ou des mollusques stérilisés ou stérilisés en fonction du sexe, ainsi que les géniteurs eux-même.
PCT/US2024/027303 2023-05-02 2024-05-01 Procédé de génération de descendance stérile et monosexe Ceased WO2024229156A2 (fr)

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CN202480045293.7A CN121712387A (zh) 2023-05-02 2024-05-01 产生不育和单性子代的方法
KR1020257040229A KR20260034684A (ko) 2023-05-02 2024-05-01 불임 및 단성 자손을 생산하는 방법
AU2024266019A AU2024266019A1 (en) 2023-05-02 2024-05-01 A method of generating sterile and monosex progeny
MX2025013109A MX2025013109A (es) 2023-05-02 2024-05-01 Un método para generar progenie estéril y monosexo
EP24800536.5A EP4704566A2 (fr) 2023-05-02 2024-05-01 Procédé de génération de descendance stérile et monosexe
IL324291A IL324291A (en) 2023-05-02 2025-10-28 A method of generating sterile and monosex progeny

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