+

WO2018013759A1 - Sélection du sexe de la volaille et de la progéniture - Google Patents

Sélection du sexe de la volaille et de la progéniture Download PDF

Info

Publication number
WO2018013759A1
WO2018013759A1 PCT/US2017/041848 US2017041848W WO2018013759A1 WO 2018013759 A1 WO2018013759 A1 WO 2018013759A1 US 2017041848 W US2017041848 W US 2017041848W WO 2018013759 A1 WO2018013759 A1 WO 2018013759A1
Authority
WO
WIPO (PCT)
Prior art keywords
bird
chromosome
cassette
gene
endogenous
Prior art date
Application number
PCT/US2017/041848
Other languages
English (en)
Inventor
Alan Blake
Richard Crockett
Aidas Nasevicius
Original Assignee
Yorktown Technologies, L.P.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yorktown Technologies, L.P. filed Critical Yorktown Technologies, L.P.
Publication of WO2018013759A1 publication Critical patent/WO2018013759A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • 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
    • A01K2207/05—Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
    • 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
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • 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
    • A01K2217/203—Animal model comprising inducible/conditional expression system, e.g. hormones, tet
    • 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
    • A01K2227/30—Bird
    • 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

Definitions

  • the present disclosure relates generally to the field of molecular biology and genetics, with implications for animal welfare. More particularly, it concems development of and methods for using New Breeding Techniques (NBTs) in avian species, such as chickens.
  • NBTs New Breeding Techniques
  • New Breeding Techniques could represent an attractive alternative or companion to conventional livestock breeding, as it may decrease the need for euthanasia and increase animal welfare.
  • non-transgenic avian species such as chickens and turkeys, has applications in biotechnology as well as for the production of meat and eggs in the agricultural industry.
  • a previous method of germ line transformation in avian species is based on a recombinant lentivirus carrying a transgene that is injected into the blastoderm (McGrew et al, 2004) or early stage chick embryo and transducing the primordial germ cells (PGCs) to produce a germline transgenic chick (Sun et al, 2012).
  • Another approach involves the isolation, in vitro culture, modification by transfection, and re-injection of PGCs to produce germ line transformed chickens (Van de Lizate et al, 2006).
  • a non-viral method is preferred for perceived biosafety and market reasons.
  • female birds comprising at least one inhibitory nucleic acid that selectively represses the expression of an endogenous developmentally critical gene, wherein the viable hatched offspring produced by crossing the female bird with a native breeder male bird are selectively female.
  • the female birds further comprise a W-chromosome rescue cassette encoding an ectopic copy of the developmentally critical gene that is not repressed by the at least one inhibitory nucleic acid, said W-chromosome rescue cassette being integrated on the heterogametic chromosome, which is the W chromosome (e.g., in the case of poultry, such as turkeys and chickens).
  • the inhibitory nucleic acid encodes a small interfering RNA (siRNA), a short hairpin RNA (shRNA) or micro RNA (miRNA).
  • the female bird is further defined as comprising: (a) an expressible autosomal repressor cassette, which is integrated in at least one copy of an autosome, said autosomal repressor cassette comprising at least one expressible inhibitory RNA that represses the expression of an endogenous developmentally critical gene; and (b) a W-chromosome ectopic rescue cassette, which is integrated in the W chromosome, said W- chromosome ectopic rescue cassette encoding the developmentally critical gene, wherein said expression cassette has a 3' UTR different from the 3' UTR of the endogenous gene.
  • the female bird further comprises an expressible W-chromosome repressor cassette, which is integrated on the W chromosome, said W-chromosome repressor cassette comprising at least one expressible inhibitory RNA that lacks a transcribed target gene in said female bird.
  • the autosomal repressor cassette is under the control of an autologous promoter, such as, for example, a copy of the endogenous promoter of the developmentally critical gene.
  • An autologous promoter may also be the promoter of a different endogenous developmentally critical gene.
  • the autosomal repressor cassette is under the control of a heterologous promoter, such as, for example, an inducible promoter, a constitutive promoter, a spatially-regulated promoter, a temporally- regulated promoter, or a synthetic promoter.
  • a heterologous promoter such as, for example, an inducible promoter, a constitutive promoter, a spatially-regulated promoter, a temporally- regulated promoter, or a synthetic promoter.
  • the W-chromosome ectopic rescue cassette is under the control of an autologous promoter, such as, for example, a copy of the endogenous promoter of the developmentally critical gene.
  • An autologous promoter may also be the promoter of a different endogenous developmentally critical gene.
  • the W-chromosome ectopic rescue cassette is under the control of a heterologous promoter, such as, for example, an inducible promoter, a constitutive promoter, a spatially-regulated promoter, a temporally- regulated promoter, or a synthetic promoter.
  • the W-chromosome repression cassette is under the control of an autologous promoter, such as, for example, a copy of the endogenous promoter of the developmentally critical gene.
  • An autologous promoter may also be the promoter of a different endogenous developmentally critical gene.
  • the W-chromosome repression cassette is under the control of a heterologous promoter, such as, for example, an inducible promoter, a constitutive promoter, a spatially-regulated promoter, a temporally- regulated promoter, or a synthetic promoter.
  • the at least one inhibitory RNA of the W-chromosome repressor cassette is located within an intron of the W-chromosome ectopic rescue cassette.
  • the W-chromosome repressor cassette is under the control of a second copy of the endogenous promoter of the developmentally critical gene.
  • the W- chromosome repressor cassette is under the control of the same promoter as the W- chromosome ectopic rescue cassette and the repressor cassette and ectopic rescue cassette are in a single polycistron.
  • the autosomal repressor cassette and the endogenous developmentally critical gene are both located on the same autosome.
  • the autosomal repressor cassette is integrated within an intron of the endogenous developmentally critical gene or in the autosome within 5 kb, 20 kb, 50 kb, or 100-200 kb of the endogenous developmentally critical gene.
  • the autosomal repressor cassette is integrated in the autosome within a gene-less region, wherein the gene-less region is the closest gene-less region to the endogenous developmentally critical gene.
  • the autosomal repressor cassette is integrated within an autosome and wherein the endogenous developmentally critical gene is located on the Z chromosome.
  • only one copy of the autosome comprises an integrated autosomal repressor cassette.
  • the female bird further comprises an expressible W-chromosome repressor cassette, which is integrated on the W chromosome, said W-chromosome repressor cassette comprising at least one expressible inhibitory RNA that lacks a transcribed target gene in said female bird.
  • the female bird produces both male and female viable offspring when bred with a native breeder line.
  • both copies of the autosome comprise an integrated autosomal repressor cassette.
  • the autosomal repressor cassette is integrated within 5 kb, 20 kb, 50 kb, or 100-200 kb of the endogenous developmentally critical gene on both copies of the autosome.
  • the autosomal repressor cassette is integrated within a gene-less region on both copies of the autosome, wherein the gene-less region is the closest gene-less region to the endogenous developmentally critical gene.
  • viable hatched offspring the female bird produces when bred with a native breeder line are selectively female offspring.
  • the bird further comprises an expressible W- chromosome repressor cassette, which is integrated on the W chromosome, said W- chromosome repressor cassette comprising at least one expressible inhibitory RNA that lacks a transcribed target gene in said female bird.
  • the autosomal repressor cassette is integrated within an autosome and wherein the endogenous developmentally critical gene is located on the Z chromosome.
  • the W-chromosome ectopic rescue cassette is integrated at a position on the chicken W chromosome (based on chicken genome assembly GalgaW (GCA 000002315.2), release 84) selected from the group consisting of approximately 235 kb - 250 kb, approximately 450 kb - 500 kb, approximately 575 kb - 615 kb, approximately 815 kb - 930 kb, and approximately 965 kb - 975 kb.
  • the W-chromosome repressor cassette is integrated within the same site on the W chromosome as the W-chromosome ectopic rescue cassette.
  • the sequence of the W-chromosome repressor cassette is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% chicken-derived.
  • the sequence of the autosomal repressor cassette is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% chicken-derived.
  • the sequence of the W-chromosome ectopic rescue cassette is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% chicken-derived.
  • the bird is a chicken, such as, for example, a layer chicken.
  • male birds are provided that comprise a Z-chromosome integrated rescue cassette encoding an ectopic copy of a developmentally critical gene that is not repressed by any inhibitory RNA, which when bred to a female bird of the present embodiments, produces both male and female viable offspring
  • male birds comprise (a) an expressible autosomal repressor cassette, which is integrated on both copies of an autosome, said autosomal repressor cassette comprising at least one expressible inhibitory RNA that represses the expression of an endogenous developmentally critical gene; and (b) a Z- chromosome ectopic rescue cassette, which is integrated on the Z chromosome, said Z- chromosome ectopic rescue cassette encoding the developmentally critical gene, wherein said Z-chromosome ectopic rescue cassette has a 3' UTR different from the 3' UTR of the endogenous gene.
  • the bird is a chicken.
  • the inhibitory RNA encodes a small interfering RNA (siRNA), a short hairpin RNA (shRNA) or micro RNA (miRNA).
  • the male bird further comprises an expressible Z- chromosome repressor cassette, which is integrated on the Z chromosome, said Z- chromosome repressor cassette comprising at least one expressible inhibitory RNA that lacks a transcribed target gene in said male bird.
  • the at least one inhibitory RNA of the Z-chromosome repressor cassette is located within an intron of the Z-chromosome ectopic rescue cassette.
  • the Z-chromosome repressor cassette is under the control of a second copy of the endogenous promoter of the developmentally critical gene.
  • the Z-chromosome repressor cassette is under the control of the same promoter as the Z-chromosome ectopic rescue cassette and the repressor cassette and ectopic rescue cassette are in a single polycistron.
  • the autosomal repressor cassette is under the control of an autologous promoter, such as, for example, a copy of the endogenous promoter of the developmentally critical gene.
  • An autologous promoter may also be the promoter of a different endogenous developmentally critical gene.
  • the autosomal repressor cassette is under the control of a heterologous promoter, such as, for example, an inducible promoter, a constitutive promoter, a spatially-regulated promoter, a temporally- regulated promoter, or a synthetic promoter.
  • a heterologous promoter such as, for example, an inducible promoter, a constitutive promoter, a spatially-regulated promoter, a temporally- regulated promoter, or a synthetic promoter.
  • the Z-chromosome ectopic rescue cassette is under the control of an autologous promoter, such as, for example, a copy of the endogenous promoter of the developmentally critical gene.
  • An autologous promoter may also be the promoter of a different endogenous developmentally critical gene.
  • the Z-chromosome ectopic rescue cassette is under the control of a heterologous promoter, such as, for example, an inducible promoter, a constitutive promoter, a spatially-regulated promoter, a temporally- regulated promoter, or a synthetic promoter.
  • the Z-chromosome repression cassette is under the control of an autologous promoter, such as, for example, a copy of the endogenous promoter of the developmentally critical gene.
  • An autologous promoter may also be the promoter of a different endogenous developmentally critical gene.
  • the Z-chromosome repression cassette is under the control of a heterologous promoter, such as, for example, an inducible promoter, a constitutive promoter, a spatially-regulated promoter, a temporally- regulated promoter, or a synthetic promoter.
  • the autosomal repressor cassette and the endogenous developmentally critical gene are both located on the same autosome.
  • the autosomal repressor cassette is integrated in the autosome within 5 kb, 20 kb, 50 kb, or 100- 200 kb of the endogenous developmentally critical gene.
  • the autosomal repressor cassette is integrated in the autosome within a gene-less region, wherein the gene- less region is the closest gene-less region to the endogenous developmentally critical gene.
  • the autosomal repressor cassette is integrated within an autosome and wherein the endogenous developmentally critical gene is located on the Z chromosome.
  • the Z-chromosome ectopic rescue cassette is integrated in a gene-less region of the Z chromosome. In some aspects, the Z-chromosome repressor cassette is integrated within the same region of the Z chromosome as the Z-chromosome ectopic rescue cassette.
  • the sequence of the Z-chromosome repressor cassette is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% chicken-derived. In some aspects, the sequence of the autosomal repressor cassette is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% chicken-derived. In some aspects, the sequence of the Z-chromosome ectopic rescue cassette is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% chicken-derived.
  • the bird is a chicken.
  • the inhibitory nucleic acid encodes a small interfering RNA (siRNA), a short hairpin RNA (shRNA) or micro RNA (miRNA).
  • methods of selective producing female progeny birds comprising (a) obtaining a female bird according to the present embodiments, said female bird comprising (i) an expressible autosomal repressor cassette, which is integrated in both copies of an autosome, said autosomal repressor cassette comprising at least one expressible inhibitory RNA that represses the expression of an endogenous developmentally critical gene, wherein said repressor cassette is under the control of a copy of the endogenous promoter of the developmentally critical gene; and (ii) a W-chromosome ectopic rescue cassette, which is integrated in the W chromosome, said W- chromosome ectopic rescue cassette encoding the developmentally critical gene, wherein said expression cassette is under the control of a copy of the endogenous promoter of the developmentally critical gene and has a 3' UTR different from the 3' UTR of the endogenous gene; and (b) breeding the obtained female bird with a native male bird,
  • the proportion of total male : female eggs produced by said breeding is not altered relative to the proportion produced when breeding two native birds, but the viable hatched offspring is selectively female. In some aspects, the proportion is not altered by more than 10%, more than 15%, more than 20%, more than 25%, or more than 30%.
  • the male progeny -containing eggs are non-viable at hatching. In some aspects, the male progeny-containing eggs are non-viable before the time of laying. In some aspects, the male progeny-containing eggs are non-viable within about 1-6 hours of laying.
  • female progeny birds are provided, wherein the female progeny chicken is produced according to a method of the present embodiment.
  • progeny of a female bird are provided, wherein the female bird produces both male and female viable offspring when bred with a native breeder line, said progeny comprising only native autosomes and comprising a W-chromosome ectopic rescue cassette, which is integrated in the W chromosome.
  • progeny of a female bird are provided, wherein the female bird produces both male and female viable offspring when bred with a native breeder line, said progeny comprising only native sex chromosomes and comprising an expressible autosomal repressor cassette, which is integrated in at least one copy of an autosome.
  • methods of maintaining a line of male and female progeny birds comprising (a) obtaining a female bird according to the present embodiments, said female bird comprises (i) an expressible autosomal repressor cassette, which is integrated in both copies of an autosome, said autosomal repressor cassette comprising at least one expressible inhibitory RNA that represses the expression of an endogenous developmentally critical gene, wherein said repressor cassette is under the control of a copy of the endogenous promoter of the developmentally critical gene; (ii) a W- chromosome ectopic rescue cassette, which is integrated in the W chromosome, said W- chromosome ectopic rescue cassette encoding the developmentally critical gene, wherein said expression cassette is under the control of a copy of the endogenous promoter of the developmentally critical gene and has a 3' UTR different from the 3' UTR of the endogenous gene; and (iii) comprises an expressible W-
  • the W-chromosome repressor cassette in the female bird comprises at least one expressible inhibitory RNA that represses the Z-chromosome ectopic rescue cassette of the male bird and the Z-chromosome repressor cassette in the male bird comprises at least one expressible inhibitory RNA that repressed the W-chromosome ectopic rescue cassette of the female bird.
  • the progeny bird is a chicken.
  • methods of breeding birds to prevent hatching of viable male offspring comprising (1) the repression of an endogenous developmentally critical gene in both male and female offspring and (2) the ectopic rescue of expression of the developmentally critical gene selectively in female offspring.
  • bird cells comprising (a) an expressible autosomal repressor cassette, which is integrated in at least one copy of an autosome, said autosomal repressor cassette comprising at least one expressible inhibitory RNA that represses the expression of an endogenous developmentally critical gene; and either (bl) a W-chromosome ectopic rescue cassette, which is integrated in the W chromosome, said W-chromosome ectopic rescue cassette encoding the developmentally critical gene, wherein said expression cassette has a 3' UTR different from the 3' UTR of the endogenous gene or (b2) a Z-chromosome ectopic rescue cassette, which is integrated in the Z chromosome, said Z-chromosome ectopic rescue cassette encoding the developmentally critical gene, wherein said expression cassette has a 3' UTR different from the 3' UTR of the endogenous gene.
  • the cell is a pluripotent cell. In some aspects, the cell is a somatic cell.
  • methods are provided of producing a commodity poultry product comprising (a) obtaining a female bird according to the present embodiments, said female bird comprising (i) an expressible autosomal repressor cassette, which is integrated in both copies of an autosome, said autosomal repressor cassette comprising at least one expressible inhibitory RNA that represses the expression of an endogenous developmentally critical gene; and (ii) a W-chromosome ectopic rescue cassette, which is integrated in the W chromosome, said W-chromosome ectopic rescue cassette encoding the developmentally critical gene, wherein said expression cassette has a 3' UTR different from the 3' UTR of the endogenous gene; and (b) breeding the obtained female bird with a male bird having only native Z chromosomes, thereby selectively producing a female progeny bird.
  • the native Z chromosomes are defined as lacking a Z-chromosome rescue cassette.
  • methods are provided of producing a commodity poultry product comprising (a) growing a female progeny according to the present embodiments; and (b) harvesting an egg laid by the female progeny bird.
  • the eggs are produced for consumption, for vaccine production, for sale, or for research.
  • an egg produced according to the present embodiments is provided.
  • isolated nucleic acid molecules comprising (a) an ectopic developmentally critical gene under the control of a copy of an endogenous promoter of the developmentally critical gene, wherein said developmentally critical gene is expressible in avian cells and is a gene that is required for embryo development, embryo homeostasis, fertilization, gastrulation, or embryogenesis; and (b) an expressible repressor cassette comprising at least one expressible inhibitory RNA that does not repress the ectopic developmentally critical gene.
  • a host cell comprising a nucleic acid molecule of the present embodiments.
  • the nucleic acid molecule is integrated into the genome of the host cell.
  • methods are provided of selling birds comprising obtaining a female bird in accordance with the present embodiments and selling said female bird.
  • methods are provided of selling a commodity poultry product comprising obtaining a commodity poultry product in accordance with the present embodiments and selling said commodity poultry product.
  • the endogenous developmental ⁇ critical gene is a gene that is required for embryo development, embryo homeostasis, fertilization, gastrulation, or embryogenesis.
  • the endogenous developmentally critical gene is essential during embryogenesis.
  • the endogenous developmentally critical gene is essential prior to stage HH04.
  • the endogenous developmentally critical gene is essential prior to stage EGK-X.
  • the endogenous developmentally critical gene is essential prior to early embryogenesis, gastrulation, early gastrulation, mid-gastrulation, the end of gastrulation, or late embryogenesis.
  • the endogenous developmentally critical gene is essential prior to neural functioning.
  • the endogenous developmentally critical gene is essential prior to neural tube formation. In some aspects, the endogenous developmentally critical gene is essential prior to the long primitive-streak stage. In some aspects, the endogenous developmentally critical gene essential prior to pain receptor activity.
  • the endogenous developmentally critical gene is zarl, cNanog, nodal, noggin, activin A (inhibin beta A), RapGEF2, Oct4 (cPouV), Frizzled 1, cVgl (GDF3), FGF4, FoxD3, Sox2, ⁇ 3 ⁇ , EGF-CFC (Cryptic, CFC1B), crescent, c-myc (v- myc), survivin (BIRC5), Wnt 3, or Wnt8C.
  • zarl cNanog, nodal, noggin, activin A (inhibin beta A), RapGEF2, Oct4 (cPouV), Frizzled 1, cVgl (GDF3), FGF4, FoxD3, Sox2, ⁇ 3 ⁇ , EGF-CFC (Cryptic, CFC1B), crescent, c-myc (v- myc), survivin (BIRC5), Wnt 3, or Wnt8C.
  • a bird's genome consists of nucleotide sequences at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% derived from genomic sequences of the same species of bird.
  • viable hatched offspring are selectively female if the proportion of female viable hatched offspring is greater than the proportion produced from breeding native lines.
  • viable hatched offspring are selectively female if at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the viable hatched offspring are female.
  • essentially free in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts.
  • the total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%.
  • Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
  • FIG. 1 Schematic illustrating a four-way cross producing all-female commercial hens.
  • FIG. 2 Diagram showing the lethal repression and rescue strategy.
  • FIG. 3 Schematic illustrating a three-way cross producing all-female commercial hens. The letter coding corresponds to the legend provided in FIG. 4.
  • FIG. 4 Schematic illustrating a three-way cross producing all-female commercial hens and post-commercial breeding.
  • FIG. 5 Schematic illustrating a four-way cross producing all-female commercial hens. The letter coding corresponds to the legend provided in FIG. 4.
  • FIG. 6 Schematic illustrating a four- way cross producing all-female commercial hens and post-commercial breeding. The letter coding corresponds to the legend provided in FIG. 4.
  • FIG. 7 Schematic illustrating the autosomal repressor cassette targeting the endogenous developmentally critical gene.
  • FIG. 8 Schematic illustrating the W- and Z-chromosome ectopic rescue expression cassette and repressor cassette.
  • the W-chromosome repressor cassette represses the expression of the Z-chromosome ectopic rescue copy of the developmentally critical gene, if present.
  • the Z-chromosome repressor cassette represses the expression of the W-chromosome ectopic rescue copy of the developmentally critical gene, if present.
  • DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
  • Embodiments of the present disclosure provide methods for sex selection in the avian species by editing the germ line.
  • the female birds that can selectively produce female offspring.
  • the female birds include an autosomal repressor cassette integrated on at least one copy of an autosome.
  • the autosomal repressor cassette encodes at least one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more inhibitory RNA(s) that can repress the expression of an endogenous developmentally critical gene, which can be any gene that is required for early development (see FIG. 7).
  • the autosomal repressor cassette is integrated on the same autosome where the endogenous developmentally critical gene is located. More preferably, the autosomal repressor cassette is integrated on the same autosome and within the regions of the regulatory elements of the endogenous developmentally critical gene. The proximity of the repressor cassette to the endogenous developmentally critical gene is also correlated with its degree of linkage, and therefore stability of inheritance over successive generations. In some cases, this preferable zone of integration is within 100-200 kb of the endogenous developmentally critical gene, because, in part, of the accessibility of the euchromatin of the genome within that distance to transcription.
  • enhancing elements which can be located at far distances, or even on other chromosomes, usually act within this preferable zone.
  • the preferable zone of integration can be much larger, and it is preferable to have the autosomal repressor cassette integrate in the closest gene-less region to the endogenous developmentally critical gene.
  • the autosomal repressor cassette is integrated on the same autosome within 50 kb of where the endogenous developmentally critical gene is located. This preferable zone corresponds to the most common distance for the position of local regulatory elements. Still more preferably, the autosomal repressor cassette is integrated on the same autosome within 20 kb of where the endogenous developmentally critical gene is located, as this preferable zone corresponds to the most common distance of the native endogenous promoter. Even more preferably, the autosomal repressor cassette is integrated on the same autosome within 5 kb of where the endogenous developmentally critical gene is located, as many of the promoters of the endogenous developmentally critical genes approach this size. An autosomal repressor cassette within this zone is more likely to be transcriptionally available. Most preferable, the autosomal repressor cassette is integrated within one or more of the introns of the endogenous developmentally critical gene.
  • the autosomal repressor cassette targets an endogenous gene on another autosome, or even on the Z chromosome.
  • the female bird also comprises a W-chromosome ectopic rescue cassette.
  • This ectopic rescue cassette encodes a copy of the developmentally critical gene but has a 3' UTR different from the 3' UTR of the endogenous gene and different from the 3' UTR of the Z-chromosome ectopic rescue cassette described below.
  • the inhibitory RNA on the autosome cannot repress the expression of the W- chromosome ectopic rescue cassette (see FIGS. 7 and 8).
  • the W chromosome comprises a W-chromosome repressor cassette encoding at least one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more inhibitory RNA(s) that can repress the expression of the Z-chromosome ectopic rescue cassette of male chickens, as described below.
  • the W-chromosome repressor cassette and W-chromosome ectopic rescue cassette may be integrated into the W chromosome at the same site or may be integrated into the W chromosome at two discrete sites.
  • RNA-Guided Nucleases such as Cas
  • Cas9 and its variants Cpfl, c2c2, or TAL repressors, ZFNs, and other sequencing specific repressors.
  • transgenic or synthetic repressors less than 100% of the sequence may be derived from the same species.
  • the male birds that are used to propagate the female birds that can selectively produce female offspring.
  • the male birds include an autosomal repressor cassette integrated on at least one copy of an autosome.
  • the autosomal repressor cassette encodes at least one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more inhibitory RNA(s) that can repress the expression of an endogenous developmentally critical gene, which can be any gene that is required for early development.
  • the autosomal repressor cassette is integrated on the same autosome where the endogenous developmentally critical gene is located.
  • the autosomal repressor cassette is integrated on the same autosome within an intron of the developmentally critical gene or within 5 kb, 20 kb, 50 kb, or 100-200 kb of where the target developmentally critical gene is located.
  • the autosomal repressor cassette targets an endogenous gene on another autosome, or even on the Z- chromosome. Thus, repression of the endogenous developmentally critical gene disrupts development of the offspring at an early stage.
  • the male bird In order to rescue the repression of the endogenous developmentally critical gene from repression by the autosomal dominant lethal repressor, and thus allow the survival of offspring, the male bird also comprises a Z- chromosome ectopic rescue cassette integrated on the Z chromosome.
  • the Z-chromosome ectopic rescue cassette encodes a copy of the developmentally critical gene but has a 3' UTR different from the 3' UTR of the endogenous gene and from the 3' UTR of the W- chromosome ectopic rescue cassette described above.
  • the inhibitory RNA transcribed from the autosome and intended to target the endogenous developmentally critical gene cannot repress the expression of the Z-chromosome ectopic rescue cassette.
  • the Z chromosome comprises a Z-chromosome repressor cassette encoding at least one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more inhibitory RNA(s) that can repress the expression of the W-chromosome ectopic rescue cassette of female chickens, as described above.
  • the Z-chromosome repressor cassette and Z-chromosome ectopic rescue cassette may be integrated into the Z chromosome at the same site or may be integrated into the Z chromosome at two discrete sites.
  • chickens of the present disclosure are bred to maintain pure lines.
  • a sexually mature male chicken of a pure line is obtained, said chicken comprising an autosomal repressor cassette on both copies of the autosome, a Z-chromosome ectopic rescue cassette on one copy of the Z chromosome, and a Z-chromosome repressor cassette on the same copy of the Z chromosome.
  • a sexually mature female chicken of a pure line is obtained, said chicken comprising an autosomal repressor cassette on both copies of the autosome, a W-chromosome ectopic rescue cassette, and a W-chromosome repressor cassette.
  • the sexually mature female and male pure line chickens are bred to propagate only offspring having the same genotype as the parents, and thus maintain a pure line.
  • female progeny having both Z-chromosome and W-chromosome ectopic rescue and repression cassettes, as well as the male progeny lacking a Z-chromosome ectopic rescue cassette and repression cassette from both copies of the Z chromosome are non-viable. As such, only progeny having the same genotype as the parent cross are obtained.
  • female chickens of the present disclosure are bred to selectively produce female progeny.
  • the female progeny of a pure line, or the female progeny of a cross between two pure lines, are bred with a male chicken having only native autosomes and native sex chromosomes.
  • the male progeny of this cross will have one copy of the autosomal repression cassette, but lack a Z-chromosome ectopic rescue cassette and repression cassette and thus be non-viable.
  • female progeny having one copy of the autosomal repression cassette and a W-chromosome ectopic rescue cassette and repression cassette are selectively obtained. It will be understood to someone skilled in the art, that variability in the degree of repression will occur depending on the particular inhibitory RNA targets selected, the particular endogenous developmentally critical gene selected, the manner and location of incorporation of the cassettes, and other factors.
  • the selective production of one gender may not be absolute, and thus selective production of at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, or 55% of the intended gender outcome may occur in any particular case.
  • selective production of one gender may occur if the proportion of viable hatched offspring of the desired gender is greater than the proportion would be from breeding native lines.
  • the developmental period in which the selected gender will become non-viable will vary. Some targets are critical soon after fertilization, others at various points in embryogenesis, gastrulation, and other stages of the bird lifecycle. In all cases in FIGS. 4 and 6 where a viable offspring is desired (Table 1, asterisked entries labeled viable), there is only one copy of the developmentally critical gene expressed. This means that whether the developmentally critical gene expression is from a native gene or from an ectopic rescue cassette, the expression level of the developmentally critical gene is not super-physiological.
  • the developmentally critical genes are important in the homeostasis of the organism, this allows the selection of targets that are sensitive to expression levels both temporally and spatially, particularly those important in sensitive periods of development. Importantly, it also allows targets to be selected wherein the target's presence or absence, and the precise timing and manner thereof, later in the lifecycle of the bird is also important, as the ectopic rescue gene, under the control of a copy of the endogenous promoter, will mimic the natural function of the endogenous gene in the organism, while still allowing the selective production of one gender. [0072] Additionally, multiple repressor cassettes all repressing a single gene may be used with the multiple repressor cassettes being rescued by a single ectopic rescue cassette.
  • the methods and animals provided herein allow for selective production of male and female birds for in breeding, while the female birds can be outbred for production of an all-female, or mostly female, layer clutch. Importantly, the birds produced by the instant methods are not transgenic and thus are highly advantageous in the agricultural industry. I. Definitions
  • avian or “bird” as used herein refers to any species, subspecies or strain of organism of the taxonomic class ayes, such as, but not limited to, such organisms as chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and ratites including ostrich, emu and cassowary.
  • poultry refers to domesticated birds, such as those used for meat production.
  • the term includes the various known strains of Gallus gallus, or chickens, (e.g.
  • layer chicken refers to an egg-laying breed of chickens selected for optimal egg production or otherwise intended to be used for the commercial production of eggs, or lines of chickens that are progenitors of such lines.
  • hybrid line refers to a line created by crossing two or more different pure lines.
  • pure line refers to a uniform line of poultry that is relatively pure genetically because of continued inbreeding.
  • construct or “cassette” as used herein refers to a linear or circular nucleotide sequence, such as DNA, that has been assembled from more than one segment of nucleotide sequence, which have been isolated from a natural source or have been chemically synthesized or modified, or combinations thereof.
  • expression cassette is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i.e. , is under the control of a promoter.
  • An "expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.
  • a "reporter gene” refers to any gene, or its product whose purpose is to facilitate identification or measurement of the genetic composition of a cell, tissue, animal, or biological matter. Reporter genes may be visual indicators (e.g. fluorescent or pigmented proteins), or may be direct or indirect targets for antibody, histochemical, chemical, microscopial, spectrophotometric, enzymatic, or other assays.
  • reporter genes include, but are not limited to, luciferin or luciferase, lacZ, GUS, and fluorescent proteins such as AmCyanl, ZsGreenl, ZsYellowl, DsRed2, DsRed-Express, AsRed2, HcRedl, mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed-monomer, mOrange, mKO, MCitrine, Venus, Ypet, EYFP, Emerald, EGFP, GFP, CyPet, mCFPm, Cerulean, or T- Sapphire.
  • fluorescent proteins such as AmCyanl, ZsGreenl, ZsYellowl, DsRed2, DsRed-Express, AsRed2, HcRedl, mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed-monomer, mO
  • An “embryonic stem (ES) cell” is an undifferentiated, pluripotent stem cell that is obtained from an embryo in an early stage or produced by artificial means (e.g. nuclear transfer) and can give rise to any differentiated cell type in an embryo or an adult, including germ cells and gametes (e.g. sperm and eggs).
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control or “operably linked” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • promoter includes constitutive, inducible, spatially restricted, and temporally restricted promoters.
  • the term promoter also includes hybrid or chimeric promoters, which contain at least two elements (e.g. , a ligand-inducible element and developmental-specific element).
  • a chimeric promoter can comprise a regulatory sequence of one promoter and the core promoter region of a second promoter. Promoters may be endogenous, ectopic, exogenous, or synthetic.
  • a "copy of an endogenous promoter” refers to an autologous promoter that is derived solely or principally, from the same species into which it is copied, including modifications to said promoter, so long as the promoter maintains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity or identity to the native, endogenous promoter.
  • a exogenous promoter refers to a promoter
  • “Developmental” as used herein refers to the process from fertilization through embryogenesis and gastrulation to hatching.
  • Avian embryos have a large mass of yolk, the cells initially divide from the center of a disk with cleavage planes that open into the yolk, and the embryo has great ability to regulate until very late stages of development. After approximately 20,000 cells have been generated, gastrulation begins. This process generates the primitive streak which defines bilateral symmetry, and through which cells from the superficial layer (epiblast) ingress to generate two new layers of embryonic cells (mesoderm and endoderm). This period of development starts to define the three axes of the future embryo (head-tail, dorsoventral and left-right), and it is at this time that many cell fates start to be fixed.
  • autologous when used in reference to a nucleotide sequence, refers to a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity (e.g. , identity) to a sequence found in the same species of bird.
  • homologous when used in reference to a nucleotide sequence, refers to a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity or identity to a sequence found in a related species of bird.
  • heterologous when used in reference to a nucleotide sequence, refers to a nucleotide sequence that is derived from another species of organism or is synthetic.
  • a heterologous sequence derived from another species has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity or identity to a sequence found in the species from which it is derived.
  • transgene refers to a gene or genetic material that has been transferred naturally, artificially, or by any number of genetic engineering techniques, from one organism and is introduced into a different organism.
  • transgenic is defined as a genetically modified organism or its progeny that contains genetic material into which DNA from another organism, xenogenic genetic material (e.g. , laboratory-designed genes), synthetic genetic material (e.g. , genetic material not found in or derived from any organism), or any combination of the above has been artificially introduced.
  • the genetic material can be in the animal's germ-cell DNA and, thus, can be transmitted from one generation to the next.
  • transgenic does not include cisgenic in which the genetic material is derived from the same species or a species that can naturally breed with the host.
  • a copy of an endogenous promoter from the chicken genome can be used in an expression cassette of the present disclosure to produce a non-transgenic chicken.
  • Any genetic sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an endogenous sequence may be considered cisgenic.
  • NBT New Breeding Technique
  • the term "native” refers to the typical form of the chromosome or bird genome when isolated from a naturally-occurring source, such as a commercially available breeder strain.
  • the native form is the form most frequently observed in a natural population.
  • site In the context of chromosomal integration, the terms “site,” “insertion site,” “locus,” “location,” and “region” may be used interchangeably to refer to an integration site.
  • heterogametic chromosome refers to genetic material that exists differentially between the sexes of a species and associates with the heterogametic sex.
  • the W chromosome is the heterogametic chromosome
  • XY systems it is the Y chromosome.
  • a "ligand-inducible promoter” or “inducible promoter” is defined as a promoter, transcriptional regulatory element, enhancer (or a combination of such elements) that is induced or repressed by the presence or absence of biotic or abiotic factors.
  • promoters include chemically regulated promoters such as those regulated by the presence or absence of alcohol, tetracycline, doxycycline, cumate, IPTG, steroids, heterologous proteins, metal or other compounds as well as physically-regulated promoters such as those regulated by the presence or absence of light and low or high temperatures.
  • tetracycline refers a class of polycyclic polyketides and includes all natural, semi-synthetic, and synthetic derivatives including, but not limited to, oxy tetracycline, doxycycline, tigecycline, and minocycline.
  • RNA interference refers to a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of mRNA molecules.
  • RNAi can include short hairpin RNA (shRNA), small interfering RNA (siRNA) and micro RNA (miRNA).
  • shRNA short hairpin RNA
  • siRNA small interfering RNA
  • miRNA micro RNA
  • RNAi can be in the form of a cassette that is transcribed in vivo to RNA that is then processed into mature shRNA, siRNA, or miRNA for transcriptional silencing.
  • Poultry meat herein refers to any product that is obtained from the bird produced by a method of the present disclosure.
  • the product can include fresh, frozen or processed (e.g., baked, roasted, fried, brined, smoked, grilled) meat (e.g., whole, sliced, ground, sandwich meat/cold cuts, bacon, giblets), eggs, stock, bones, and any byproducts thereof.
  • the chick embryo originates in the germinal disc, a small region of cytoplasm situated at the animal pole of the ovum (the familiar yolk). During the first third of its development, the embryo remains floating at the surface of the yolk while the extraembryonic membranes grow around the yolk and become vascularized. In the remaining period of development, the embryo grows at the expense of the food reserves in the egg. Details regarding each Eyal-Giladi stage and Hamburger Hamilton stage are provided in Table 2 and can be found on the world wide web at echickatlas.org/ecap/hamburger_handlton_stages/StageDefinition/stagecriteria.htrnl. See also Sheng (2014), which is incorporated herein by reference in its entirety.
  • the albumen In the uterus, the albumen is doubled in volume by the absorption of uterine fluid (plumping fluid), and finally the shell undergoes slow calcification. For hens laying in long sequences of one egg per day, oviposition is followed within 15-30 min by the next ovulation and the cycle is repeated.
  • Embryonic morphogenesis This phase takes place in the first 3 days of incubation of the egg (stages 1-18, Hamburger and Hamilton, 1951). At stage 20, the embryo is 10 mm in length, and the extra embryonic blastoderm extends around the yolk to its equator.
  • Tail bud becomes a short, straight cone.
  • Tail bud 37-40 somites; Leg buds become slightly larger than wing buds; Tail bud
  • HH35 Webs between digits and toes are inconspicuous; Beak becomes lengthened.
  • Lower eyelid covers one-third to one half of cornea; Upper eyelid has reached
  • wing are long, tapering cones.
  • Length of beak from anterior edge of nostril to tip of bill 4.0 mm; Length of
  • Length of beak from anterior edge of nostril to tip of upper bill 4.5 mm;
  • Length of third toe 14.9 ⁇ 0.8 mm.
  • Length of third toe 16.7 ⁇ 0.8 mm.
  • Length of beak from anterior edge of nostril to tip of upper bill 5.0 mm;
  • Length of third toe 18.6 ⁇ 0.8 mm.
  • Length of beak from anterior edge of nostril to tip of upper bill 5.7 mm;
  • Length of third toe 20.4 ⁇ 0.8 mm.
  • HH45 Beak is shiny; Yolk sac half enclosed in body cavity.
  • sperm sorting is not a modality that can be employed in poultry, as the sperm of a rooster (the homogametic sex) contains spermatozoa that only contain Z- chromosomes.
  • the present disclosure relates to methods for avian sex selection by producing birds that selectively produce progeny of a particular sex when bred with certain breeder lines. Such methods involve genetically modifying the heterogametic sex (the sex that carries the two different sex chromosomes and therefore determines the sex of the offspring), such that only the gamete of the undesired gender is disabled, or will not complete embryogenesis.
  • the technology of the present disclosure has particular applicability in the field of agriculture, and particularly in the poultry industry.
  • the ability to pre-determine or direct the sex of poultry has the potential to improve both the economics and management of the industry.
  • the present disclosure provides several advantages while also overcoming the deficiencies of the prior art. After the animals of the present disclosure are created, there is no need for further technology to produce offspring of a particular sex.
  • a female giving rise to single sex offspring could be used in multiple normal matings or in artificial insemination protocols.
  • the female progeny are of high commercial value because of their non-transgenic, female nature.
  • ES cells are derived from the inner cell mass of blastocysts and have a high in vitro differentiating capability.
  • ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo, then culturing the inner mass cells on a feeder layer of non-growing cells. The replated cells can continue to proliferate and produce new colonies of ES cells, which can be removed, dissociated, replated again, and allowed to grow. This process of "subculturing" undifferentiated ES cells can be repeated a number of times to produce cell lines containing undifferentiated ES cells (U.S. Patent Nos. 5,843,780; 6,200,806; 7,029,913, incorporated herein by reference).
  • ES cells have the potential to proliferate while maintaining their pluripotency.
  • ES cells are useful in research on cells and on genes which control cell differentiation.
  • the pluripotency of ES cells combined with genetic manipulation and selection can be used for gene analysis studies in vivo via the generation of transgenic, chimeric, and knockout chickens.
  • Avian embryonic stem cells such turkey or chicken cells
  • the ES cells can be seeded on a layer of feeder cells and cultured for about 20 passages.
  • the ability to maintain ES undifferentiated cells in continuous culture enables in vitro transfection of such cells and ideally the selection of transfected cells that contain a desired genetic trait prior to their transfer to the inner cell mass of a developing embryo to generate chimeric animals. Ideally, at least some of the resultant chimeric animals will be able to segregate the DNA construct via the germ line and, hence, produce progeny carrying the desired trait(s).
  • a germ cell is any biological cell that gives rise to the gametes of an organism that reproduces sexually. In many animals, the germ cells originate in the primitive streak and migrate via the gut of an embryo to the developing gonads. There, they undergo cell division of two types, mitosis and meiosis, followed by cellular differentiation into mature gametes, either eggs or sperm.
  • PGCs Avian primordial germ cells undergo a migration through the vasculature on their path to the gonad where they become sperm or ova producing cells.
  • PGCs come from the epiblast and move to the hypoblast to form the germinal crescent (anterior extraembryonic structure).
  • the gonocytes then squeeze into blood vessels and use the circulatory system for transport. They squeeze out of the vessels when they are at height of the gonadal ridges.
  • Cell adhesion on the endothelium of the blood vessels and molecules, such as chemo-attractants, are probably involved in helping PGCs migrate.
  • avian PGCs can be isolated from chicken eggs during stage 12-14 of embryonic development and transferred to suitable cell culture medium (e.g., M199 medium). The isolated PGCs are then counted and separated manually (e.g., using a pipette). Thereafter, PGCs collected from these different avian embryos are pooled (to increase PGC numbers) and incubated in the subject growth factor containing medium.
  • suitable cell culture medium e.g., M199 medium
  • the culture medium preferably contains LIF, bFGF, SCF and IGF as well as other substituents typically comprised in PGC and embryonic stem cell medium. More specifically, the medium will preferably comprise a-MEM and additionally includes fetal calf serum, LIF, bFGF, IGF and SCF. Also, PGCs can be cultured in the absence or presence of feeder cells. Certain aspects of the present disclosure concern ex vivo manipulation of primordial germ cells (PGCs) followed by injection of the cells back into a recipient embryo.
  • PPCs primordial germ cells
  • the ZW sex-determination system determines the sex.
  • Males are the homogametic sex (ZZ), while females are the heterogametic sex (ZW). Since males can only donate a Z chromosome, it is the female gamete that determines the sex of the offspring.
  • Sex selection methods are performed by introducing an autosomal nucleic acid repressor cassette into at least one copy of an autosomal chromosome of the germ line of said animal.
  • the autosomal repressor cassette represses the expression of an endogenous developmentally critical gene, which is essential for embryogenesis and/or early development, optimally located on the same chromosome, even more optimally located on the same chromosome and within an intron of the endogenous developmentally critical gene or within 5 kb, 20 kb, 50 kb, or 100-200 kb of the endogenous developmental critical gene.
  • an ectopic rescue cassette and a second repressor cassette are integrated into the W chromosome in female birds and into one Z chromosome in male birds.
  • the W- or Z- chromosome ectopic rescue cassette encodes a copy of the endogenous developmentally critical gene; however, the expression of the W- or Z-chromosome ectopic rescue cassette is not subj ect to repression by the autosomal repressor cassette.
  • the W-chromosome repressor cassette represses the expression of the Z-chromosome ectopic rescue cassette, if present.
  • the Z-chromosome repressor cassette represses the expression of the copy of the W-chromosome ectopic rescue cassette, if present.
  • the autosomal repressor cassette interferes with the process of fertilization, completion of embryogenesis and/or homeostasis. By interfering with fertilization or embryogenesis, no progeny are produced. Progeny are only produced if the egg has one, but not both, of the edited W or Z chromosomes, since the edited sex chromosome will supply endogenous levels of the otherwise repressed autosomal gene.
  • female progeny can be selectively produced by breeding a female bird as disclosed herein with a native male bird.
  • male progeny can be produced for the purpose of line propagation by breeding a female bird as disclosed herein with a male bird as disclosed herein.
  • RNAi RNA interference
  • RNAi pathways are activated by various forms of double-stranded (ds) RNAs that contain sequences which are homologous to the mRNA transcript of a target gene.
  • ds double-stranded
  • RNAi includes small interfering RNA (siRNA), short hairpin RNA (shRNA), and micro RNA (miRNA).
  • Short hairpin RNA (shRNA) transcripts adopt a stable stem-loop structure in solution; can be easily be expressed from a cloned oligonucleotide template; and are a convenient and reproducible means of activating RNAi in cells.
  • the recombinant construct can comprise an RNAi expression cassette.
  • the expressible repression cassette can comprise the coding regions of a gene(s) that is transcribed in vivo to shRNA.
  • the shRNA oligonucleotide design usually comprises a target sense sequence (e.g. , a 19-base target sense sequence), a hairpin loop (e.g. , 7-9 nucleotides), a target antisense sequence (e.g. , a 19-base target antisense sequence) and a RNA Pol II terminator sequence.
  • the hairpin loop can be 5'-TTCAAGAGA-3' (SEQ ID NO: 66; Sui et al , 2002).
  • the RNA Pol II terminator sequence is usually a 5-6 nucleotide poly(T) tract.
  • the RNAi expression cassette may be polycistronic and express more than one shRNA.
  • the cassette can comprise several shRNA coding regions interspaced by linker sequences and followed by a terminator sequence.
  • the shRNA coding regions can encode different shRNAs, multiple copies of the same shRNA, or a combination thereof. Similarly, these may be placed outside the gene of interest, or within a single intron in the gene of interest, multiple introns, or all introns. It is preferred to avoid altering the first intron as it may contain regulatory elements, though this risk may be mitigated by comparing the native intron sequence to sequences of known regulatory function.
  • the native intron of interest with the first introns of analogous genes in other species, such as mouse, to determine if there are conserved regions, and to avoid the alteration of such regions. It is even more preferred to avoid the addition of shRNA coding sequences to native introns of less than approximately 300 bp, and preferred that the shRNA coding sequences constitute no more than an approximately 100% increase in size of the native intron.
  • the cassette additionally can comprise a minimal U6 snRNA promoter.
  • a tetracycline-inducible, RNA-Polymerase II hybrid promoter can be used comprising a modified Tet-responsive element with direct repeats of the tetO 19-mer from the tet operon to a minimal U6 snRNA promoter (Kunkel and Pederson, 1989).
  • Expression of the cassette is preferably under the control of a copy of the promoter element from the endogenous target gene of interest.
  • the repression construct can comprise an siRNA expression cassette.
  • siRNA Small interfering RNA
  • siRNA is a class of double-stranded RNA molecules about 20-25 nucleotides in length. siRNA interferes with the expression of specific genes with complementary nucleotide sequences by causing mRNA to be broken down after transcription, resulting in no translation. 3. miRNA
  • the recombinant cassette can comprise a microRNA
  • miRNA for silencing gene targets.
  • miRNA is a small non-coding RNA molecule about 22 nucleotides in length that functions in RNA silencing and post-transcriptional regulation of gene expression. It is preferred to use miRNA backbones from miRNA encoded in the introns of native genes within the organism. This will mitigate the risk of cryptic and other erroneous splices. In the case of polycistronic shRNA, it is also preferred to use different miRNA backbones for each of the cistrons to mitigate the risk of recombination among the polycistronic shRNA transcripts and promote efficient shRNA processing.
  • RNAi can target specific genes that are involved in fertilization, embryo development, embryo homeostasis, gastrulation, or embryogenesis but are not expressed in adult organisms (Table 3).
  • a developmental ⁇ critical gene has an important role in fertilization, embryo development, embryo homeostasis, gastrulation, or embryogenesis but is not required during later development.
  • the choice of target genes is not limited by concerns about super-physiologic expression resulting from redundant expression from multiple copies of the target gene present in a bird. Super-physiologic expression levels are avoided even when an ectopic rescue gene is present because the repressor cassettes only allow one copy of the target gene to be actively transcribed in any given scenario.
  • the RNAi could target important early regulators of fertilization, such as zarl (zygote arrest 1) or zarl-like genes.
  • the RNAi could target genes involved in embryogenesis (Table 3).
  • the targeted gene is expressed early in embryogenesis and not during late embryogenesis.
  • the target gene is only, or predominantly, expressed during embryogenesis.
  • the target gene's gene product is only important during embryogenesis and has no, or minimal, effect in mature birds.
  • a gene involved in embryogenesis could be Nodal which is required for multiple embryonic events, including dorso-ventral and left-right axis formation, and embryonic layer formation, is expressed zygotically, and may be contributed maternally. Generally, it is not expressed in adults.
  • Another gene involved in embryogenesis is EGF-CFC which serves as a co-receptor in nodal signaling; is contributed both maternally and zygotically; and has been demonstrated to be not essential for adult viability and fertility, as EGF-CFC null mutants can be rescued by mRNA injection and give rise to normal fertile adults.
  • RNAi an anti-apoptotic protein expressed during embryogenesis, is virtually absent in all adult tissues, and knockout embryonic lethal.
  • RapGEF2 although functioning later in embryogenesis, could also be targeted as it is required for embryonic hematopoiesis; the null mutant mutation is embryonic lethal in mice.
  • ESCs embryonic stem cells
  • ⁇ 3 ⁇ DNA cytosine-5 -methyltransferase 3 ⁇
  • a DNA methyltransferase that functions early in embryogenesis, specifically in the ICM, epiblast and embryonic ectoderm
  • Oct4 FGF4, FoxD3, Sox2
  • Nanog Nanog
  • cassettes could comprise RNAi targeted to genes involved in homeostasis.
  • Genes encoding transcription factors for the apoptotic pathway can be used as target genes (e.g. , Bax, Caspase).
  • the autosomal repression cassette comprises at least one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more inhibitory RNA(s) that can repress the expression of an endogenous developmentally critical gene.
  • the inhibitory RNA(s) are designed to target the endogenous 3' UTR of the developmentally critical gene.
  • Six exemplary inhibitory RNA target sequences for a select group of endogenous developmentally critical genes are as follows: zarl (SEQ ID NOs: 6-11), nodal (SEQ ID NOs: 12-17), Oct4 (SEQ ID NOs: 18-23), EGF-CFC (SEQ ID NOs: 24-29), and survivin (SEQ ID NOs: 30-35).
  • An inhibitory RNA target sequence may be at least 80%, 85%, 90%, 95%, or 99% identical to any of the foregoing sequences. It is envisioned that any one, two, or at least three of each set of inhibitory RNA(s) can be incorporated in an autosomal repression cassette under the control of a copy of the endogenous target gene's promoter.
  • the W-chromosome repression cassette comprises at least one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more inhibitory RNA(s) that can repress the expression of the Z-chromosome ectopic rescue cassette.
  • the inhibitory RNA(s) are designed to target the unique 3' UTR of the Z-chromosome ectopic rescue cassette, the design of which is described below.
  • the Z-chromosome repression cassette comprises at least one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more inhibitory RNA(s) that can repress the expression of the W- chromosome ectopic rescue cassette.
  • the inhibitory RNA(s) are designed to target the unique 3' UTR of the W-chromosome ectopic rescue cassette, the design of which is described below.
  • the two rescue + repression cassettes reciprocally inhibit each other such that no rescue gene expression occurs from either sex chromosome (see FIG. 2).
  • the inhibitory RNA(s) of a repression cassette may be under the control of a promoter separate from the promoter driving the expression of the ectopic rescue gene.
  • the inhibitory RNA(s) of a repression cassette may be incorporated within one or more introns of the ectopic rescue gene and thus under the control of the ectopic rescue gene's promoter.
  • Expression cassettes are employed to express a protein coding sequence, either for subsequent purification and delivery to a cell, or for use directly in a genetic-based delivery approach, such as to a bird embryo.
  • Expression requires that appropriate signals be provided in the vectors, and include various regulatory elements, such as enhancers/promoters from mammalian, avian, and/or non-mammalian/avian sources that drive expression of the ectopic developmentally critical gene in cells.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of promoters, including those for thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • the expression cassette of the present disclosure comprises a promoter for control of transcription.
  • the promoter is a region of DNA that initiates transcription of the target gene located upstream of the DNA.
  • Developmentally -regulated promoters known in art can be used in the expression cassette.
  • the promoter provides temporal or spatial control of target gene expression.
  • the promoter will be a copy of the same promoter element as the endogenous developmentally critical gene.
  • the 3' UTR for each of the W-chromosome and Z-chromosome ectopic rescue cassettes are unique from each other and unique relative to the 3' UTR of the endogenous developmentally critical gene.
  • fragments of chicken genomic DNA will be selected and inserted into the 3' UTR of the rescue gene. It is important not to truncate the 3' UTR and to include the past predicted poly-adenylation signal sequence. As such, each rescue gene will be supplemented with a unique chicken genomic DNA fragment. Guidelines for selection of fragments of chicken genomic DNA for such use are as follows.
  • Preferred fragments will be selected from a large chromosome, such as chicken chromosomes 1-5, preferably chicken chromosome 1.
  • Preferred fragments will be selected from a chromosomal region not overlapping the centromere.
  • the chromosomal region is preferably a gene desert, i.e., no predicted or known genes are located within 100 kb and preferably within 1,000 kb.
  • the chromosomal region preferably contains no repeats, SNPs, or indels.
  • the chromosomal region should be between 35% and 55% GC.
  • This region is away from the centromere, which is located around position 74 MB; is located approximately 100 kb away from the nearest gene; contains several sequences free of repeats; contains several sequences free to SNPs or indels; and is between approximately 25%-50% GC.
  • the cassettes can be integrated in insertion sites on the autosome and W or Z chromosome by multiple methods. These include, but are not limited to, endonucleases such as the CPJSPR/Cas9 system, TALENs, ZFNs, or Meganucleases, and self-editing technologies such as transposases systems, Cre-LoxP or FLP-FRT.
  • endonucleases such as the CPJSPR/Cas9 system, TALENs, ZFNs, or Meganucleases
  • self-editing technologies such as transposases systems, Cre-LoxP or FLP-FRT.
  • the cassette be integrated into the same autosome where the endogenous developmentally critical gene is located.
  • the cassette will be integrated into the autosome within 50 kb of the endogenous developmentally critical gene.
  • the cassette will be integrated in a gene-less region of the autosome that is closest to the endogenous developmentally critical gene.
  • insertion sites on the chicken W chromosome are several preferred insertion sites on the chicken W chromosome (NCBI Reference Sequence: NC_006126.4) for the introduced cassette.
  • Preferred insertion sites are extragenic material lacking transposons, long-terminal repeats, or other repeated elements.
  • Exemplary integration positions on the a position on the chicken W chromosome include approximately 235 kb - 250 kb, 450 kb - 500 kb, 575 kb - 615 kb, 815 kb - 930 kb, and 965 kb - 975 kb (using the numbering based on the chicken assembly, obtained on the world wide web at useast.ensembl.org/Gallus_gallus/Info/Index, incorporated herein by reference).
  • NCBI Reference Sequence: NC_006127.4 is much less restricted. As such, insertion will preferably occur in any gene-less region, with the exception of regions proximal to the telomeres (2 Mb terminal regions) and the centromere (located at position 42.22 - 42.26 Mb).
  • CRISPRs Clustered regularly interspaced short palindromic repeats
  • CRISPRs are segments of DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposure to virus.
  • CRISPRs are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with cas genes that code for proteins related to CRISPRs.
  • the CRISPR/Cas9 system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPR spacers recognize and silence these exogenous genetic elements like RNAi in eukaryotic organisms.
  • SRSR Short Regularly Spaced Repeats
  • Cas9 is a nuclease, an enzyme specialized for cutting DNA with two active cutting sites, HNH and RuvC, one for each strand of the DNA's double helix.
  • CRISPR was first shown to work as a genome engineering/editing tool in human cell culture by 2012 It has since been used in a wide range of organisms including (e.g.) bakers yeast (S. cerevisiae), zebrafish, nematodes (C. elegans), plants, mice, and several other organisms. Additionally CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes. Libraries of tens of thousands of guide RNAs are now available.
  • CRISPR repeats range in size from 24 to 48 base pairs. They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic. Repeats are separated by spacers of similar length. Some CRISPR spacer sequences exactly match sequences from plasmids and phages, although some spacers match the prokaryote's genome (self-targeting spacers). New spacers can be added rapidly in response to phage infection.
  • CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than forty different Cas protein families had been described. Of these protein families, Casl appears to be ubiquitous among different CRISPR/Cas systems. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat- associated mysterious proteins (RAMPs). More than one CRISPR subtype may occur in a single genome.
  • RAMPs repeat- associated mysterious proteins
  • Exogenous DNA is apparently processed by proteins encoded by Cas genes into small elements (-30 base pairs in length), which are then somehow inserted into the CRISPR locus near the leader sequence.
  • RNAs from the CRISPR loci are constitutively expressed and are processed by Cas proteins to small RNAs composed of individual, exogenously-derived sequence elements with a flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level.
  • Evidence suggests functional diversity among CRISPR subtypes.
  • the Cse (Cas subtype E. coli) proteins (called CasA-E in E. coli) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains.
  • Cas6 processes the CRISPR transcripts.
  • CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Casl and Cas2.
  • the Cmr (Cas RAMP module) proteins found in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs.
  • RNA-guided CRISPR enzymes are classified as type V restriction enzymes.
  • Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. One or both sites can be disabled while preserving Cas9's ability to home located its target DNA.
  • tracrRNA and spacer RNA can be combined into a "single-guide RNA" molecule that, mixed with Cas9, can find and cut the correct DNA targets. Jinek et al. proposed that such synthetic guide RNAs might be able to be used for gene editing.
  • Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Cas9-mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts.
  • Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas9- associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence.
  • scaRNA CRISPR/Cas9- associated RNA
  • Cas9 requires a short RNA to direct the recognition of DNA targets (Mali et al. , 2013a). Though Cas9 preferentially interrogates DNA sequences containing a PAM sequence NGG it can bind here without a protospacer target. However, the Cas9-gRNA complex requires a close match to the gRNA to create a double strand break (Cho et al., 2013; Hsu et al. , 2013). CRISPR sequences in bacteria are expressed in multiple RNAs and then processed to create guide strands for RNA (Bikard et al, 2013).
  • gRNA RNA polymerase type III promoter U6 (Mali et al , 2013).
  • Synthetic gRNAs are slightly over 100 bp at the minimum length and contain a portion which targets the 20 protospacer nucleotides immediately preceding the PAM sequence NGG; gRNAs do not contain a PAM sequence.
  • the CRISPR Type II system uses RNA-guided endonuclease technology for genome engineering.
  • the two distinct components of the system include a guide RNA and an endonuclease Cas9 (CRISPR associated nuclease).
  • the guide RNA is a combination of the endogenous bacterial crRNA and tracrRNA into a single chimeric guide RNA (gRNA) transcript.
  • gRNA chimeric guide RNA
  • the gRNA combines the targeting specificity of crRNA with the scaffolding properties of the tracRNA into a single transcript.
  • the genomic target sequence can be modified or permanently disrupted.
  • the gRNA/Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement to the target sequence in the genomic DNA.
  • the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence.
  • PAM Protospacer Adjacent Motif
  • the binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the wild-type Cas9 can cut both strands of DNA causing a Double Strand Break (DSB). Cas9 will cut 3-4 nucleotides upstream of the PAM sequence.
  • a DSB can be repaired through one of two general repair pathways: (1) the Non-Homologous End Joining (NHEJ) DNA repair pathway or (2) the Homology Directed Repair (HDR) pathway.
  • NHEJ Non-Homologous End Joining
  • HDR Homology Directed Repair
  • the NHEJ repair pathway often results in inserts/deletions (InDels) at the DSB site that can lead to frameshifts and/or premature stop codons, effectively disrupting the open reading frame (ORF) of the targeted gene.
  • the HDR pathway requires the presence of a repair template, which is used to fix the DSB. HDR faithfully copies the sequence of the repair template to the cut target sequence. Specific nucleotide changes can be introduced into a targeted gene by the use of HDR with a repair template.
  • the Cas9 nuclease has two functional domains: RuvC and HNH, each cutting a different DNA strand. When both of these domains are active, the Cas9 causes double strand breaks (DSBs) in the genomic DNA. In the absence of a suitable repair template, the DSB is repaired by the Non-Homologous End Joining (NHEJ) DNA repair pathway. During NHEJ repair, InDels (insertions/deletions) may occur as a small number of nucleotides are either inserted or deleted at random at the DSB site. InDels alter the Open Reading Frame (ORF) of the target gene, which may significantly change the amino acid sequence downstream of the DSB.
  • NHEJ Non-Homologous End Joining
  • InDels could also introduce a premature stop codon either by creating one at the DSB or by shifting the reading frame to create one downstream of the DSB. Any of these outcomes of the NHEJ repair pathway can be leveraged by scientists to disrupt their target gene. It is important to note that the InDels induced by NHEJ will be random, so the type and extent of gene disruption will need to be determined experimentally.
  • the CRISPR/Cas9 system has a short recognition sequence and can tolerate multiple mismatches, resulting in a low specificity and leading to off-target DNA digestion.
  • "Nickase” a mutated form of Cas9 protein with only one DNA-cutting domain, was constructed (Ran et al, 2013).
  • Nickase creates a single strand DNA cut - a nick, instead of a double-strand DNA break.
  • Each Nickase has its own target site, and when two nicks in proximity occur, a double strand DNA break is formed. An off- target single strand nick is efficiently repaired and DNA sequence is not changed.
  • the requirement of two Nickases cutting different DNA strands in proximity dramatically increases CRISPR/Cas9 system specificity.
  • Nickase is preferentially used in genome editing when a high specificity is desired.
  • the dCas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme (that retains its gRNA-binding ability) to known regulatory domains.
  • the transcriptional activator VP64 when fused to dCas9, is capable of up-regulating gene transcription of targeted genes to enhance expression.
  • the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.
  • Fusions of dCas9 with transcriptional repressors, such as KRAB have been developed to further improve the silencing capabilities of the CRISPR system. By choosing a gRNA binding site near the promoter region of a gene, researchers can artificially activate or repress the transcription of a gene.
  • TALENs TALENs
  • Transcription activator-like effector nucleases can also be used for integration of a cassette as disclosed herein.
  • TALENs are artificial restriction enzymes generated by fusing a TAL effector DNA-binding domain to a DNA cleavage domain.
  • Transcription activator-like effectors TALEs
  • TALEs Transcription activator-like effectors
  • restriction enzymes can be engineered that are specific for any desired DNA sequence.
  • these restriction enzymes are introduced into cells, they can be used for genome editing in situ, a technique known as genome editing with engineered nucleases.
  • TALEs may be used without the cleavage domain to knockdown gene targets (Zhang, 2014).
  • ZFNs zinc-finger nucleases
  • Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes.
  • Custom-designed ZFNs that combine the non-specific cleavage domain (N) of Fokl endonuclease with zinc-finger proteins (ZFPs) offer a general way to deliver a site-specific DSB to the genome, and stimulate local homologous recombination by several orders of magnitude.
  • ZFPs zinc-finger proteins
  • meganucleases are used to integrate a cassette of the present disclosure.
  • Meganucleases are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs); as a result this site generally occurs only once in any given genome.
  • the 18-base pair sequence recognized by the I-Scel meganuclease would on average require a genome twenty times the size of the human genome to be found once by chance (although sequences with a single mismatch occur about three times per human-sized genome).
  • Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes.
  • Meganucleases are "molecular DNA scissors" that can be used to replace, eliminate or modify sequences in a highly targeted way. By modifying their recognition sequence through protein engineering, the targeted sequence can be changed.
  • the cassette is integrated through the use of transposase systems.
  • a transposable element (TE or transposon) is a DNA sequence that can change its position within the genome, sometimes creating or reversing mutations and altering the cell's genome size.
  • Transposons can be used in genetic research and recombinant genetic engineering for insertional mutagenesis. Insertional mutagenesis is when transposons function as vectors to help remove and integrate genetic sequences. Given their relatively simple design and inherent ability to move DNA sequences, transposons are highly compatible at transducing genetic material, making them ideal genetic tools.
  • transposase systems include Sleeping Beauty, miniTol2 and PiggyBac. a. miniTol2
  • a miniTol2 plasmid system (Balciunas et al, 2006) may be used for integration of the cassette.
  • one plasmid contains the terminal Tol2 sequences flanking the promoter driving the reporter gene and the other plasmid (pTrans) contains the transposase sequence under the control of another promoter.
  • Promoters for use in the miniTol2 plasmid system can be the pCAGGS promoter, the CMV IE promoter.
  • the miniTol-reporter gene sequence is incorporated into the target genomic DNA while the pTrans sequence will not be incorporated.
  • the PiggyBac transposon which is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism.
  • the Super PB transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and moves the contents from the original sites and efficiently integrates them into TTAA chromosomal sites.
  • ITRs inverted terminal repeat sequences
  • the expression construct is introduced by DNA microinjection, viral transduction, vesicle fusion, electroporation, or chemical transfection.
  • Cell-based transfer may involve primordial germ cells or embryonic stem cells.
  • vectors may be directly injected into an avian fetus (optionally with a lipid- based transfection reagent), thereby transfecting stem or germline cells in the organism in vivo (see, e.g., Tyack et al , 2013, incorporated herein by reference).
  • transfection reagents can include lipofectamine such as Lipofectamine 2000 CD complex.
  • the plasmids and transfection reagent can be combined at a plasmid:plasmid ratio of 1 :2 and transfection reagent: plasmid ratio of 3 ⁇ : 1.8 ⁇ g.
  • lipofectamine 2000 is its potential cytotoxicity in the presence of antibiotics.
  • transfection reagent alternatives suitable for in vivo transformation, including, but not limited to:
  • the cassette can be introduced by DNA injection (in vivo) or microinjection (ex vivo).
  • DNA injection the nucleic acids are injected (e.g. , into the aorta) of a developing bird, optionally with a transfection or transduction reagent.
  • DNA microinjection is a simple mechanical process usually involving an inverted microscope with a magnification power of around 200x (though sometimes it is performed using a dissecting stereo microscope at 10-50x or a traditional compound upright microscope at similar power to an inverted model).
  • the target cell is positioned under the microscope and two micromanipulators— one holding the pipette and one holding a microcapillary needle usually between 0.5 to 5 ⁇ in diameter (larger if injecting stem cells into an embryo)— are used to penetrate the cell membrane and/or the nuclear envelope. In this way the process can be used to introduce a vector into a single cell.
  • the expression vector may be introduced in vitro such as into embryonic stem cells, primordial germ cells (PGCs) or zygotes.
  • PSCs primordial germ cells
  • zygotes after injection of the construct into zygotes, in vitro differentiated blastocysts can be explanted into culture to derive ES cells.
  • the expression vector is introduced by DNA microinjection in vivo to the embryo (e.g., PGCs) of the animal.
  • Pronuclear injection is a technique used to modify organisms by injecting genetic material into the nucleus of a fertilized oocyte.
  • These animals can be generated by pronuclear or cytoplasmic injection of DNA.
  • the RNA and DNA can be simultaneously be injected into the cytoplasm, simultaneously be injected into the pronucleus, or the Cas9 and sgRNA can be injected into the cytoplasm with pronuclear injection of the DNA vector.
  • the RNA and DNA are simultaneously injected into the cytoplasm or nucleus to achieve the targeted insertion. V. Examples
  • Plasmids The pT7-Cas9-Nickase plasmid (Origene catalog number
  • GE100020 SEQ ID NO: 36
  • GE100025 a mutated "Nickase” Cas9 gene
  • gRNA Cloning Vector pT7-Guide-IVT plasmid Origene catalog number GE100025; SEQ ID NO: 37
  • gRNA pairs were designed using the Feng Zhang Target Finder (on the world- wide-web at crispr.mit.edu) to target a region in chicken chromosome 22 (chromosome 22 [RefSeq: NC_006109.4], 2,255,653:2,255,855, antisense strand [SEQ ID NO: 38]) in order to insert an autosomal repressor cassette into the first intron of the endogenous Nodal gene.
  • the sequences of the gRNAs pair targets including PAM sequences, are provided in Table 4. Table 4.
  • W chromosome [RefSeq: NC_006126.4], 884,712:884,961, sense strand [SEQ ID NO: 45]) in order to insert a W-chromosome ectopic rescue cassette that contains a Z-chromosome repression cassette within the first intron of the rescue gene.
  • the sequences of the gRNAs pair targets, including PAM sequences, are provided in Table 5.
  • Z chromosome [RefSeq: NC_006127.4], 30,028,551 - 30,028,800, sense strand [SEQ ID NO: 50]) Z chromosome ectopic rescue cassette that contains a W- chromosome repression cassette within the first intron of the rescue gene.
  • the sequences of the gRNAs pair targets, including PAM sequences, are provided in Table 6.
  • gRNAs Cloning of gRNAs is performed according to CRISPR plasmid instructions (available on the world-wide-web at origene.com/assets/documents/CRISPR- CAS9/CRISPR_manual.pdf, which is incorporated herein by reference in its entirety).
  • An alternative method to produce the gRNA templates is to generate them using PCR. Briefly, GE10025 is digested with BsmBI as the template, and primers T7-target (SEQ ID NO. 57: taatacgactcactatagggNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNgtm where the desired
  • gRNA_3'common SEQ ID NO. 58: CAAGCTTTTTAAAAGCACCGACTCGGT.
  • the PCR product is used as a template for gRNA sequence using T7 polymerase. Other polymerases can be used, such as SP6. In this instance, the 5' end of the T7-target primer would have to be changed accordingly to include the correct promoter sequence.
  • the autosomal repressor cassette that targets endogenous Nodal see FIG. 7
  • the W-chromosome rescue cassette and W-chromosome repressor cassette that targets the Z-chromosome rescue cassette see FIG.
  • the Z-chromosome rescue cassette and Z-chromosome repressor cassette that targets the W-chromosome rescue cassette are each individually cloned into a cloning vector.
  • the cloning vector, with the vector backbone removed, is used as the HDR template and mixed with Cas9 Nickase mRNA or protein and gRNAs for the transformation.
  • the two cloning vectors, one that contains the autosomal repressor cassette that targets endogenous Nodal and a second that contains the W-chromosome rescue cassette and W-chromosome repressor cassette are used in combination.
  • the two cloning vectors one that contains the autosomal repressor cassette that targets endogenous Nodal and a second that contains the Z-chromosome rescue cassette and Z- chromosome repressor cassette are used in combination.
  • Linearized pT7-Cas9-Nickase plasmid is used as the template for in vitro transcription using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England BioLabs, catalog number E2050S), supplemented with 3'-0-Me-m7G(5')ppp(5')G RNA Cap Structure Analog (New England BioLabs, catalog number S1411S).
  • a poly (A) tail is added using E. coli Poly (A) Polymerase (New England BioLabs, catalog number M0276S).
  • Linearized pT7-Guide-IVT plasnuds with inserted gRNA templates are used as the templates for in vitro transcription using the HiScribe T7 Quick High Yield RNA Synthesis Kit.
  • the synthesized Cas9 Nickase mRNA and gRNAs are purified according to the HiScribe T7 Quick High Yield RNA Synthesis Kit manual (available on the world-wide- web at neb.com/ ⁇ /media/Catalog/All-
  • RNA is measured by optical density at 260 nm.
  • PGCs of chicken embryos are directly transfected in vivo with a formulation of lipid transfection reagent, such as, lipofectamine 2000, Cas9/gRNA, and cloning vector with the plasmid backbone removed.
  • the embryos are microinj ected with the DNA and RNAs by cutting a window in the pointed side of a recipient egg to allow access to the stage 14 HH embryo (Tyack et al, 2013, incorporated herein by reference for all purposes).
  • 1-2 of transfection complex is injected into the dorsal aorta using a pulled glass micropipette.
  • PGCs including those transformed as described, migrate to the germinal crest, and contribute to the germline, forming offspring capable of passing on the edited gene to subsequent generations. Survival percentages from this technique can vary with rates of less than 1%, l%-5%, 5%-10%, 10%-25%, 25%-50%, or in some applications, rates greater than 50% may be achieved with this method, and it avoids several of the challenges inherent in PGC culture. It is important to note that with respect to the current disclosure, even a single surviving offspring is sufficient. Ova from ED 7 and 14 are dissected and PCR-analyzed for the presence of the expressible repressor cassette and the W or Z chromosome expression and repressor cassette.
  • Chickens are grown to sexual maturity and quantitative real time PCR
  • qPCR qPCR is used to detect the presence of the inserted DNA in female and male chickens either in ova (or, alternatively, by tissue sampling prior to sexual maturity).
  • the desired female chickens will have the autosomal repressor cassette inserted into both strands of chromosome 22 and the W-chromosome ectopic rescue cassette and repressor cassette that targets the Z chromosome copy of Nodal inserted on the W chromosome.
  • the desired male chickens will have the autosomal repressor cassette inserted into both strands of chromosome 22 and the Z- chromosome ectopic rescue expression cassette and repressor cassette that targets the W chromosome copy of Nodal inserted on one copy of the Z chromosome.
  • Chickens identified as positive from the qPCR screen are mated with each other and the offspring are PCR- analyzed to confirm stable integration of the inserted DNA into transfected PGCs and germ- line transmission of the inserted DNA to the offspring.
  • the only resulting viable chickens should have the autosomal repressor cassette inserted into both strands of chromosome 22 as well as either the Z-chromosome ectopic rescue cassette and repressor cassette that targets the W chromosome copy of Nodal inserted on one copy of the Z chromosome or the W-chromosome ectopic rescue cassette and repressor cassette that targets the Z chromosome copy of Nodal inserted on the W chromosome.
  • Plasmids The pT7-Cas9-Nickase plasmid (Origene catalog number GE100020; SEQ ID NO: 36) containing a mutated "Nickase" Cas9 gene and the gRNA Cloning Vector pT7-Guide-IVT plasmid (Origene catalog number GE100025; SEQ ID NO: 37) containing the backbone of gRNA were purchased from Origene.
  • gRNA pairs were designed using the Feng Zhang Target Finder (on the world- wide-web at crispr.mit.edu) to target a region in chicken chromosome 17 (chromosome 17 [RefSeq: NC_006104.4], 458,762:458,899, antisense strand [SEQ ID NO: 59]) in order to insert an autosomal repressor cassette downstream of the endogenous cPouV gene.
  • the sequences of the gRNAs pair targets including PAM sequences, are provided in Table 7.
  • W chromosome ectopic rescue cassette contains a Z-chromosome repression cassette downstream of the ectopic rescue cassette.
  • the elements of the cassette may be arranged as follows: (cPouv promoter)-(cPouv coding region)-(cPouv 3'UTR + chromosome-specific inhibitory target sequence)-(cPouv promoter)-(shRNA cassette)-(cPouv 3'UTR).
  • the sequences of the gRNAs pair targets, including PAM sequences, are provided in Table 8. Table 8.
  • Z chromosome [RefSeq: NC_006127.4], 30,028,551 - 30,028,800, sense strand [SEQ ID NO: 50]) in order to insert a Z-chromosome ectopic rescue cassette that contains a W- chromosome repression cassette downstream of the ectopic rescue cassette.
  • the elements of the cassette may be arranged as follows: (cPouv promoter)-(cPouv coding region)-(cPouv 3'UTR with chromosome-specific inhibitory target sequence)-(cPouv promoter)-(shRNA cassette)-(cPouv 3'UTR with autosomal repression targets removed).
  • the sequences of the gRNAs pair targets, including PAM sequences, are provided in Table 9.
  • Cloning of gRNAs is performed according to CRISPR plasmid instructions (available on the world-wide-web at origene.com/assets/documents/CRISPR- CAS9/CRISPR_manual.pdf, which is incorporated herein by reference in its entirety).
  • An alternative method to produce the gRNA templates is to generate them using PCR. Briefly, GE10025 is digested with BsmBI as the template, and primers T7-target (SEQ ID NO. 57: taatacgactcactatagggNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNgtttt where the desired 16-20 base guide sequence is inserted) and gRNA_3'common (SEQ ID NO.
  • the PCR product is used as a template for gRNA sequence using T7 polymerase. Other polymerases can be used, such as SP6. In this instance, the 5' end of the T7-target primer would have to be changed accordingly to include the correct promoter sequence.
  • the autosomal repressor cassette that targets endogenous cPouV see FIG. 7
  • the W-chromosome rescue cassette and W-chromosome repressor cassette that targets the Z-chromosome ectopic rescue cassette see FIG. 8
  • the Z- chromosome ectopic rescue cassette and Z-chromosome repressor cassette that targets the W- chromosome ectopic rescue cassette see FIG.
  • the vector backbone may include one or more antibiotic resistance markers or other selection markers, along with other origins of replication and regulatory elements, multiple- cloning sites, and the like.
  • the cloning vector, with the vector backbone removed, is used as the HDR template and mixed with Cas9 Nickase mRNA or protein and gRNAs for the transformation.
  • the two cloning vectors one that contains the autosomal repressor cassette that targets endogenous cPouV and a second that contains the W-chromosome rescue cassette and W-chromosome repressor cassette are used in combination.
  • the two cloning vectors one that contains the autosomal repressor cassette that targets endogenous cPouV and a second that contains the Z-chromosome rescue cassette and Z-chromosome repressor cassette are used in combination.
  • pT7-Cas9-nickase and pT7-Guide-IVT plasmids are optimized for in vitro RNA synthesis and encode T7 RNA polymerase promoter.
  • the plasmids are linearized according to the manufacturer's instructions.
  • Linearized pT7-Cas9-Nickase plasmid is used as the template for in vitro transcription using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England BioLabs, catalog number E2050S), supplemented with 3'-0-Me-m7G(5')ppp(5')G RNA Cap Structure Analog (New England BioLabs, catalog number S1411S). After the transcription, a poly(A) tail is added using E. coli Poly(A) Polymerase (New England BioLabs, catalog number M0276S).
  • RNA molecules Linearized pT7-Guide-IVT plasmids with inserted gRNA templates are used as the templates for in vitro transcription using the HiScribe T7 Quick High Yield RNA Synthesis Kit.
  • the synthesized Cas9 Nickase mRNA and gRNAs are purified according to the HiScribe T7 Quick High Yield RNA Synthesis Kit manual (available on the world-wide- web at neb.com/ ⁇ /media/Catalog/All- Products/9D7209047C8E4B34ABE47B635EB600E2/Datacards%20or%20Manuals/manualE 2050.pdf, which is incorporated herein by reference in its entirety), and dissolved in nuclease- free water.
  • the concentration of RNA is measured by optical density at 260 nm.
  • PGCs of chicken embryos are directly transfected in vivo with a formulation of lipid transfection reagent, such as, lipofectamine 2000, Cas9/gRNA, and cloning vector with the plasmid backbone removed.
  • the embryos are microinj ected with the DNA and RNAs by cutting a window in the pointed side of a recipient egg to allow access to the stage 14 HH embryo (Tyack et al , 2013, incorporated herein by reference for all purposes).
  • a micropipette 1-2 ⁇ of transfection complex is injected into the dorsal aorta using a pulled glass micropipette.
  • PGCs including those transformed as described, migrate to the germinal crest, and contribute to the germline, forming offspring capable of passing on the edited gene to subsequent generations. Survival percentages from this technique can vary with rates of less than 1%, l%-5%, 5%-10%, 10%-25%, 25%-50%, or in some applications, rates greater than 50% may be achieved with this method, and it avoids several of the challenges inherent in PGC culture. It is important to note that with respect to the current disclosure, even a single surviving offspring is sufficient. Ova from ED 7 and 14 are dissected and PCR-analyzed for the presence of the expressible repressor cassette and the W or Z chromosome expression and repressor cassette.
  • Chickens are grown to sexual maturity and quantitative real time PCR (qPCR) is used to detect the presence of the inserted DNA in female and male chickens either in ova (or, alternatively, by tissue sampling prior to sexual maturity).
  • the desired female chickens will have the autosomal repressor cassette inserted into both strands of chromosome 17 and the W-chromosome ectopic rescue cassette and repressor cassette that targets the Z chromosome copy of cPouV inserted on the W chromosome.
  • the desired male chickens will have the autosomal repressor cassette inserted into both strands of chromosome 17 and the Z- chromosome ectopic rescue expression cassette and repressor cassette that targets the W chromosome copy of cPouV inserted on one copy of the Z chromosome.
  • Chickens identified as positive from the qPCR screen are mated with each other and the offspring are PCR- analyzed to confirm stable integration of the inserted DNA into transfected PGCs and germ- line transmission of the inserted DNA to the offspring.
  • the only resulting viable chickens should have the autosomal repressor cassette inserted into both strands of chromosome 17 as well as either the Z-chromosome ectopic rescue cassette and repressor cassette that targets the W chromosome copy of cPouV inserted on one copy of the Z chromosome or the W-chromosome ectopic rescue cassette and repressor cassette that targets the Z chromosome copy of cPouV inserted on the W chromosome.
  • the method of in vivo transfection of PGCs with the DNA constructs generates stable germ-line male and female chickens capable of passing the inserted DNA onto the next generation.
  • a sexually mature male chicken of a pure line comprising an autosomal repressor cassette, which represses the expression of an endogenous developmentally critical gene, on both copies of the autosome where the endogenous developmentally critical gene is located. Since repression of the endogenous developmentally critical gene results in an autosomal dominant lethal, the male chicken also comprises an ectopic rescue cassette on one copy of the Z chromosome that encodes an ectopic copy of the developmentally critical gene that is not repressed by the autosomal repression cassette. The same Z chromosome that comprises the ectopic rescue cassette also comprises a second repressor cassette for which no target gene is present in the male chicken.
  • the second repressor cassette represses the expression of the ectopic rescue cassette integrated on the W chromosome of the female chicken described below.
  • a sexually mature female chicken of a pure line is obtained, said chicken comprising an autosomal repressor cassette, which represses the expression of an endogenous developmentally critical gene, on both copies of the autosome where the endogenous target gene of interest is located. Since repression of the endogenous developmentally critical gene results in an autosomal dominant lethal, the female chicken also comprises an ectopic rescue cassette on the W chromosome that encodes an ectopic copy of the endogenous developmentally critical gene that is not repressed by the repression cassette integrated in the autosomes.
  • the W chromosome also comprises a second repressor cassette for which no target gene is present in the female chicken.
  • the second repressor cassette represses the expression of the ectopic rescue cassette integrated on the Z chromosome of the male chicken described above.
  • the sexually mature female and male pure line chickens are bred to propagate only offspring having the same repressor/rescue cassette genotype as the parents, and thus maintain a pure line.
  • the female progeny having an ectopic rescue cassette and both Z- and W-chromosome repression cassettes as well as the male progeny lacking an ectopic rescue cassette and repression cassette from both copies of the Z chromosome are non-viable. As such, only progeny having the same genotype as the parent cross are obtained.
  • a female progeny of the above cross is bred with a male chicken having only native autosomes and native sex chromosomes.
  • the male progeny will lack an ectopic rescue cassette and second repression cassette and thus be non-viable.
  • only female progeny having one autosomal copy of the autosomal repression cassette and an ectopic rescue cassette and second repression cassette on the W chromosome are obtained.
  • the right panel of FIG. 4 if these female progeny are further crossed with a male chicken having only native autosomes and native sex chromosomes, then both male and female progeny will be produced. As such, post- commercial breeding can be recognized.
  • the male chickens also comprises an ectopic rescue cassette on one copy of the Z chromosome that encodes a copy of the developmentally critical gene that is not repressed by the repression cassette integrated in the autosomes.
  • the same Z chromosome that comprises the ectopic rescue cassette also comprises a second repressor cassette for which no target gene is present in the male chickens. The second repressor cassette represses the expression of the ectopic rescue cassette integrated on the W chromosome of the female chickens described below.
  • one sexually mature female chicken of both a first and second pure line is obtained, said chicken comprising a first repressor cassette, which represses the expression of an endogenous developmentally critical gene, on both copies of the autosome where the endogenous developmentally critical gene is located.
  • the female chickens Since repression of the endogenous developmentally critical gene results in an autosomal dominant lethal, the female chickens also comprises an ectopic rescue cassette on the W chromosome that encodes a copy of the developmentally critical gene that is not repressed by the repression cassette integrated in the autosomes.
  • the W chromosome also comprises a second repressor cassette for which no target gene is present in the female chickens. The second repressor cassette represses the expression of the ectopic rescue cassette integrated on the Z chromosome of the male chickens described above.
  • a sexually mature female from the cross of one of the two pure lines, as described above, is bred with a sexually mature male from the other of the two pure lines.
  • the female progeny having an ectopic rescue cassette and second repression cassette on both the Z and W chromosomes as well as the male progeny lacking an ectopic rescue cassette and second repression cassette from both copies of the Z chromosome are non-viable.
  • progeny having the same genotype as the grandparent cross are obtained, except that the progeny are no longer a pure line but rather a hybrid line.
  • a female progeny of the above cross is bred with a male chicken having only native autosomes and native sex chromosomes.
  • the male progeny will lack an ectopic rescue cassette and second repression cassette and thus be non-viable.
  • only female progeny having one autosomal copy of the first repression cassette and an ectopic rescue cassette and second repression cassette on the W chromosome are obtained.
  • the bottom right panel of FIG. 6 if these female progeny are further crossed with a male chicken having only native autosomes and native sex chromosomes, then both male and female progeny will be produced. As such, post-commercial breeding can be easily recognized.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Environmental Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des poulets produisant sélectivement une progéniture femelle. Selon certains aspects, l'invention concerne un oiseau ou des cellules de ce dernier qui comprennent une cassette de répresseur autosomique intégrée sur au moins une copie d'un autosome, qui peut supprimer l'expression d'une protéine essentielle au développement précoce. Selon certains aspects, l'invention concerne un oiseau ou des cellules de ce dernier qui comprennent une cassette de sauvetage ectopique et une cassette de répresseur sur le chromosome W ou Z, qui peut sélectivement sauver le développement d'embryons chez les animaux de descendance. L'invention concerne également des procédés de production de tels animaux et de production d'œufs de volaille alimentaires.
PCT/US2017/041848 2016-07-13 2017-07-13 Sélection du sexe de la volaille et de la progéniture WO2018013759A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662361814P 2016-07-13 2016-07-13
US62/361,814 2016-07-13

Publications (1)

Publication Number Publication Date
WO2018013759A1 true WO2018013759A1 (fr) 2018-01-18

Family

ID=60951919

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/041848 WO2018013759A1 (fr) 2016-07-13 2017-07-13 Sélection du sexe de la volaille et de la progéniture

Country Status (1)

Country Link
WO (1) WO2018013759A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111073963A (zh) * 2019-12-10 2020-04-28 南京农业大学 一种一日龄鉴别鸡受精蛋性别的方法
WO2020178822A1 (fr) * 2019-03-05 2020-09-10 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Oiseaux à édition de génome
CN111903609A (zh) * 2020-08-13 2020-11-10 江苏京海禽业集团有限公司 一种具有屠体标识高产青脚优质肉鸡的制种方法
CN113424794A (zh) * 2021-04-16 2021-09-24 安徽农业大学 一种优质抗病型地方鸡新品系选育方法及应用
CN114015705A (zh) * 2021-11-28 2022-02-08 华中科技大学同济医学院附属协和医院 一种小鼠体外受精繁育性别选择方法
CN116019061A (zh) * 2023-03-07 2023-04-28 江苏省家禽科学研究所 一种中快速屠宰型优质鸡的培育方法
CN117546801A (zh) * 2023-12-07 2024-02-13 广州大学 一种遗传育种的全雄性大刺鳅高效繁育方法及应用
WO2024059699A3 (fr) * 2022-09-16 2024-05-16 Joseph Fenton Lawler Procédés de trans-épissage et compositions pour la génération d'une descendance de sexe unique
JP7493194B1 (ja) 2023-05-11 2024-05-31 株式会社セツロテック 鳥類の雌雄判別方法、鳥類、生産方法、および卵の集団
WO2024249570A3 (fr) * 2023-06-02 2025-03-06 New York University Poulet à sexe unique avec système toxine-antitoxine

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010027569A1 (en) * 2000-02-15 2001-10-04 Tim Cantrell Method of fertilizing an avian egg in the shell
US20120046263A1 (en) * 2009-03-02 2012-02-23 Univeristy of Georgia Research Method to alter sex ratios in avian offspring
US20120084873A1 (en) * 2009-02-08 2012-04-05 Andrew Sinclair Sex-determination and methods of specifying same
US20120227119A1 (en) * 2008-12-17 2012-09-06 Australian Poultry Crc Pty Ltd Methods of Modulating the Sex of Avians
US20130288364A1 (en) * 2010-10-28 2013-10-31 Whitehead Institute For Biomedical Research Engineered kinetochores and uses thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010027569A1 (en) * 2000-02-15 2001-10-04 Tim Cantrell Method of fertilizing an avian egg in the shell
US20120227119A1 (en) * 2008-12-17 2012-09-06 Australian Poultry Crc Pty Ltd Methods of Modulating the Sex of Avians
US20120084873A1 (en) * 2009-02-08 2012-04-05 Andrew Sinclair Sex-determination and methods of specifying same
US20120046263A1 (en) * 2009-03-02 2012-02-23 Univeristy of Georgia Research Method to alter sex ratios in avian offspring
US20130288364A1 (en) * 2010-10-28 2013-10-31 Whitehead Institute For Biomedical Research Engineered kinetochores and uses thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GUIOLI ET AL.: "PITX2 controls asymmetric gonadal development in both sexes of the chick and can rescue the degeneration of the right ovary", DEVELOPMENT, vol. 134, 24 October 2007 (2007-10-24), pages 4199 - 4208, XP055452834 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7667087B2 (ja) 2019-03-05 2025-04-22 ザ ステイト オブ イスラエル ミニストリー オブ アグリカルチャー アンド ルーラル ディベロップメント アグリカルチュラル リサーチ オーガニゼイション （エー．アール．オー．） （ボルカニ インスティテュート） ゲノム編集された鳥
WO2020178822A1 (fr) * 2019-03-05 2020-09-10 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Oiseaux à édition de génome
CN113692225A (zh) * 2019-03-05 2021-11-23 以色列农业和农村发展部农业研究组织(范卡尼中心) 经基因组编辑的鸟类
CN111073963A (zh) * 2019-12-10 2020-04-28 南京农业大学 一种一日龄鉴别鸡受精蛋性别的方法
CN111073963B (zh) * 2019-12-10 2023-04-14 南京农业大学 一种一日龄鉴别鸡受精蛋性别的方法
CN111903609A (zh) * 2020-08-13 2020-11-10 江苏京海禽业集团有限公司 一种具有屠体标识高产青脚优质肉鸡的制种方法
CN111903609B (zh) * 2020-08-13 2021-09-07 江苏京海禽业集团有限公司 一种具有屠体标识高产青脚优质肉鸡的制种方法
CN113424794A (zh) * 2021-04-16 2021-09-24 安徽农业大学 一种优质抗病型地方鸡新品系选育方法及应用
CN114015705A (zh) * 2021-11-28 2022-02-08 华中科技大学同济医学院附属协和医院 一种小鼠体外受精繁育性别选择方法
WO2024059699A3 (fr) * 2022-09-16 2024-05-16 Joseph Fenton Lawler Procédés de trans-épissage et compositions pour la génération d'une descendance de sexe unique
CN116019061B (zh) * 2023-03-07 2023-07-07 江苏省家禽科学研究所 一种中快速屠宰型优质鸡的培育方法
CN116019061A (zh) * 2023-03-07 2023-04-28 江苏省家禽科学研究所 一种中快速屠宰型优质鸡的培育方法
JP7493194B1 (ja) 2023-05-11 2024-05-31 株式会社セツロテック 鳥類の雌雄判別方法、鳥類、生産方法、および卵の集団
WO2024249570A3 (fr) * 2023-06-02 2025-03-06 New York University Poulet à sexe unique avec système toxine-antitoxine
CN117546801A (zh) * 2023-12-07 2024-02-13 广州大学 一种遗传育种的全雄性大刺鳅高效繁育方法及应用

Similar Documents

Publication Publication Date Title
US12213466B2 (en) Process for using CRISPR to transfect primordial germ cells in avians
WO2018013759A1 (fr) Sélection du sexe de la volaille et de la progéniture
Woodcock et al. Gene editing in birds takes flight
US20170152526A1 (en) Engineering of humanized car t-cell and platelets by genetic complementation
KR20170003585A (ko) 돼지에서 멀티플렉스 유전자 편집
CN105934512A (zh) 用于在核基因转移的受体中产生没有发育能力的卵子的方法和组合物
EP3367785A2 (fr) Compositions et procédés de production d'organe assisté par un embryon chimère
WO2020198541A1 (fr) Porcs résistants au virus du syndrome dysgénésique et respiratoire du porc (prrsv)
KR20180128386A (ko) 유전적 상보성에 의한 인간화 신장의 조작
US20210037797A1 (en) Inducible disease models methods of making them and use in tissue complementation
US20200149063A1 (en) Methods for gender determination and selection of avian embryos in unhatched eggs
Barkova et al. Innovative Approaches to Genome Editing in Chickens
WO2021171688A1 (fr) Procédé de knock-in de gène, procédé de production d'une cellule à knock-in de gène, cellule à knock-in de gène, procédé d'évaluation du risque de cancer, procédé de production de cellules cancéreuses, et kit pour leur utilisation
Kolluri EXPRESSION ANALYSIS OF GROWTH REGULATORY GENES AND OPTIMIZATION OF CRISPR/Cas9 MEDIATED GENE EDITING OF ACTIVIN RECEPTOR TYPE 2B (ACVR2B) IN BROILER CHICKEN Thesis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17828435

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205 DATED 02.05.2019)

122 Ep: pct application non-entry in european phase

Ref document number: 17828435

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载