Genetic complementary compositions and methods

Abstract

In several exemplary embodiments herein, germline complementation methods and compositions, in particular NANOS3 deficient cells and non-human animals, are described. In some embodiments, the NANOS3 deficient non-human animal is germline ablated.

Description

Cross-referencing related applications

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 327,168, filed April 4, 2022, entitled “Genetic Complementation Compositions and Method,” which is incorporated herein by reference in its entirety. sequence list

[0002] This application contains a sequence list created on April 4, 2023, and consisting of 66,156 bytes, submitted electronically as an XML file named 081906-129621_ST26.xml. The contents of the sequence list are incorporated herein by reference in their entirety. Technical Field

[0003] The topics disclosed in this article generally concern non-human animals undergoing genetic phylogenetic ablation and their uses. Background Technology

[0004] Conventional genetic selection and breeding programs have generated elite genetic seed reserves. However, there is a lag in genetic improvement between these elite nuclear seed reserves and commercial animals. This lag is due to the generational intervals in conventional breeding programs. Therefore, methods are needed to increase the rate of genetic improvement, particularly at the commercial animal level.

[0005] References to or identification of any document in this application are not an admission that such document is available as prior art to this invention. Summary of the Invention

[0006] Some example embodiments of this document describe engineered non-human animal cells or populations thereof containing NANOS3 gene modifications, wherein the NANOS3 gene modifications reduce or eliminate the expression of the NANOS3 gene product.

[0007] In some example embodiments, the NANOS3 gene modification is the insertion of one or more nucleotides; the deletion of one or more nucleotides; the substitution of one or more nucleotides; or any combination thereof. In some example embodiments, the NANOS3 gene modification is located in exon 1 of the NANOS3 gene, optionally in the zinc finger domain of the NANOS3 gene.

[0008] In some example embodiments, the engineered non-human animal cells or populations thereof are bovine, horse, pig, sheep, goat, camel, deer, dog, cat, mouse, rabbit, or guinea pig cells.

[0009] In some example embodiments, one or both NANOS3 alleles are modified. In some example embodiments, the engineered non-human animal cells or populations thereof are monoallelic for the NANOS3 gene modification. In some example embodiments, the engineered non-human animal cells or populations thereof are bialelic for the NANOS3 gene modification. In some example embodiments, the engineered non-human animal cell populations do not express a functional NANOS3 gene or gene product.

[0010] In some example embodiments, the engineered non-human animal cells are heterozygous or homozygous for the NANOS3 gene modification, wherein the NANOS gene modification is optionally NANOS3 gene knockout.

[0011] In some example embodiments, the engineered non-human animal cells or populations thereof are engineered male cells or populations thereof. In some example embodiments, the engineered non-human animal cells are engineered female cells or populations of cells.

[0012] In some example embodiments, the engineered nonhuman animal cells or populations thereof are engineered somatic cells or populations thereof. In some example embodiments, the engineered nonhuman animal cells or populations thereof are engineered germ cells or populations thereof. In some example embodiments, the engineered germ cells or populations thereof are engineered gametes or populations thereof. In some example embodiments, the engineered gametes or populations thereof are engineered sperm or populations thereof or engineered oocytes or populations thereof. In some example embodiments, the engineered germ cells or populations thereof are engineered immature germ cells or populations thereof. In some example embodiments, the engineered immature germ cells or populations thereof are engineered sperm cells or populations thereof or engineered oocytes or populations thereof. In some example embodiments, the engineered nonhuman animal cells are populations of their engineered embryonic cells, optionally wherein the engineered embryonic cells are fertilized eggs. In some example embodiments, wherein the populations of their engineered nonhuman animal cells are engineered blastocyst cells or populations thereof, optionally engineered inner cell mass cells or populations thereof. In some example embodiments, the engineered nonhuman animal cells or populations thereof are engineered stem cells or populations thereof, optionally engineered embryonic stem cells or populations thereof, or induced pluripotent stem cells or populations thereof. In some example embodiments, the engineered nonhuman animal cells or cell populations are engineered spermatogonial stem cells or populations thereof, or engineered oogonial stem cells or populations thereof. In some example embodiments, the engineered nonhuman animal cells or cell populations are primordial germ cells or populations thereof, or engineered primordial germ cell-like cells or populations thereof. In some example embodiments, the engineered nonhuman animal cells or populations thereof are engineered self-renewing cells or populations thereof. In some example embodiments, the engineered nonhuman animal cells are pluripotent, totipotent, or multipotent.

[0013] Some example embodiments of this document describe engineered nonhuman animals, embryos, or their offspring, which comprise engineered nonhuman animal cells or populations thereof as described in any of the foregoing paragraphs or elsewhere herein.

[0014] In some example embodiments, the engineered nonhuman animal, embryo, or its offspring is a chimera. In some example embodiments, the engineered nonhuman animal, embryo, or its offspring is a mosaic. In some example embodiments, the engineered nonhuman animal, embryo, or its offspring is not a chimera. In some example embodiments, the engineered nonhuman animal, embryo, or its offspring is not a mosaic. In some example embodiments, at least 0.0001% to 100% of at least one cell or all cells of the engineered nonhuman animal, embryo, or its offspring are any of the preceding paragraphs or engineered nonhuman animal cells as described elsewhere herein.

[0015] In some example embodiments, the engineered non-human animal, embryo, or its offspring is male. In some example embodiments, the engineered non-human animal, embryo, or its offspring is female. In some example embodiments, the engineered non-human animal is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, rat, rabbit, or guinea pig.

[0016] In some example embodiments, the engineered nonhuman animal, embryo, or its offspring further comprises a second cell population including one or more cells, wherein the second cell population does not contain any of the preceding paragraphs or engineered nonhuman animal cells as described elsewhere herein, and wherein the second cell population is germline-capable cells, germ cells, or gametes. In some example embodiments, the second cell population comprises or consists of: one or more embryonic cells, optionally fertilized eggs or inner cell mass cells; stem cells, optionally embryonic stem cells or induced pluripotent stem cells; spermatogonial stem cells or oogonial stem cells; primordial germ cells; or primordial germ cell-like cells. In some example embodiments, the second cell population comprises or consists of one or more sperm cells or one or more oocytes. In some example embodiments, the second cell population comprises or consists of sperm or eggs. In some example embodiments, the second cell population comprises or consists of one or more engineered cells, wherein the one or more engineered cells contain one or more genetic modifications in one or more target genes, and wherein the one or more target genes are not NANOS3. In some example embodiments, the second cell population does not contain or consists of engineered cells or a population thereof. In some example embodiments, the second cell population comprises an elite genome, a genome-selected genome, or both or composed of the latter.

[0017] In some exemplary embodiments herein, complementary nonhuman animals or embryos are described, the complementary nonhuman animals or embryos comprising or consisting of a first cell population including one or more cells and a second cell population including one or more cells, wherein the first cell population consists of any of the preceding paragraphs and / or engineered nonhuman animal cells or populations thereof as described elsewhere herein, wherein the second cell population is not any of the preceding paragraphs and / or engineered nonhuman cells or populations thereof as described elsewhere herein.

[0018] In some example embodiments, the second cell population comprises or is composed of one or more engineered cell populations, said one or more engineered cell populations containing one or more genetic modifications in one or more target genes, wherein said one or more target genes are not NANOS3. In some example embodiments, the second cell population is not engineered cells or a population thereof. In some example embodiments, the second cell population comprises an elite genome, a genome-selected genome, or both. In some example embodiments, the second cell population comprises or is composed of: one or more embryonic cells, optionally fertilized eggs or inner cell mass cells; stem cells, optionally embryonic stem cells or induced pluripotent stem cells; spermatogonial stem cells or oogonial stem cells; primordial germ cells; or primordial germ cell-like cells. In some example embodiments, the second cell population is self-renewing cells. In some example embodiments, the second cell population is pluripotent, totipotent, or multipotent. In some example embodiments, the second cell population is germline capable.

[0019] In some example embodiments, the complementary embryo is a preimplantation embryo, optionally a fertilized egg, a 2-cell, 4-cell, 8-cell, 16-cell, blastocyst, or morula. In some example embodiments, the first cell population represents a percentage of the complementary nonhuman animal or embryo's cells in the range of about 25% to at most but not including 100%. In some example embodiments, the complementary nonhuman animal or embryo contains at least one cell from the second cell population, optionally wherein the second cell population represents a percentage of the engineered nonhuman animal or embryo's cells in the range of any non-zero percentage to about 75%. In some example embodiments, the complementary embryo is a day 3 post-fertilization embryo, a day 4 post-fertilization embryo, a day 5 post-fertilization embryo, or a day 6 post-fertilization embryo. In some example embodiments, the complementary embryo on day 3 post-fertilization contains about 5 cells from the second cell population; the complementary embryo on day 4 post-fertilization contains about 5 cells from the second cell population; the complementary embryo on day 5 post-fertilization contains about 8-10 cells from the second cell population; and the complementary embryo on day 6 post-fertilization contains about 10-20 cells from the second cell population.

[0020] In some example embodiments, the complementary embryo is a morula. In some example embodiments, the complementary non-human animal or embryo is male. In some example embodiments, the complementary non-human animal or embryo is female. In some example embodiments, the complementary non-human animal or embryo is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, mouse, hare, or guinea pig.

[0021] Some example embodiments of this document describe non-human animals that develop or arise from any of the preceding paragraphs or complementary non-human animals or embryos as described elsewhere herein. In some example embodiments, one or more germ cells of the engineered animal are derived from the second cell population. In some example embodiments, approximately 0.001% to 100% of the germ cells are derived from the second cell population. In some example embodiments, the non-human animal is male. In some example embodiments, the non-human animal is female.

[0022] Some example embodiments of this document describe any of the preceding paragraphs or one or more complementary non-human animals or offspring of non-human animals as described elsewhere in this document.

[0023] Some example embodiments describe methods for generating non-human animals or embryos modified with NANOS3, the methods comprising introducing one or more NANOS3 gene modifications into non-human animal cells, wherein the NANOS3 gene modification reduces or eliminates the expression of the NANOS3 gene product; and one or more of the following techniques: somatic cell nuclear transfer, oocyte pronuclear DNA microinjection, fertilized egg microinjection or embryo microinjection, intracytoplasmic sperm injection, in vitro fertilization, embryo transfer, in vitro embryo culture, or any combination thereof.

[0024] In some example embodiments, the NANOS3 gene modification is the insertion of one or more nucleotides; the deletion of one or more nucleotides; the substitution of one or more nucleotides; or any combination thereof. In some example embodiments, the NANOS3 gene modification is located in exon 1 of the NANOS3 gene, optionally in the zinc finger domain of the NANOS3 gene.

[0025] In some example embodiments, one or both NANOS3 alleles are modified. In some example embodiments, the non-human animal or embryo is monoallelic for the NANOS3 gene modification. In some example embodiments, the non-human animal or embryo is bialelic for the NANOS3 gene modification. In some example embodiments, the engineered non-human animal or embryo does not express a functional NANOS3 gene or gene product. In some example embodiments, the non-human animal or embryo is heterozygous or homozygous NANOS3 gene knockout. In some example embodiments, the non-human animal or embryo is germline ablation.

[0026] In some example embodiments, the non-human animal or embryo is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, rat, hare, or guinea pig. In some example embodiments, the non-human animal or embryo is male. In some example embodiments, the non-human animal or embryo is female.

[0027] In some example embodiments, introducing one or more NANOS3 gene modifications into the non-human animal cells includes CRISPR-Cas-mediated gene modification, zinc finger nuclease gene modification, TALEN-mediated gene modification, recombinase-mediated gene modification, leader editing-mediated gene modification, broad-spectrum nuclease-mediated gene modification, transposase / transposon-mediated gene modification, or any combination thereof.

[0028] In some example embodiments, introducing one or more NANOS3 gene modifications into the non-human animal cells involves using a CRISPR-Cas system, wherein the guide RNA of the CRISPR-Cas system targets exon 1 of the NANOS3 gene, optionally in the zinc finger region, and optionally selected from any one or any combination thereof of SEQ ID NO:39-45.

[0029] Some example embodiments of this document describe a method for complementing nonhuman animal embryos, the method comprising introducing a self-renewing exogenous cell population into a nonhuman animal preimplantation embryo, optionally around day 3, 4, 5, or 6 post-fertilization; optionally washing the nonhuman animal preimplantation embryo in HEPES or other suitable buffer; and culturing the nonhuman preimplantation embryo in a suitable culture medium, optionally consisting of a suitable bovine medium supplemented with at least N2, B27, FGF, and IWR-1 in a 1:1 volume ratio.

[0030] In some example embodiments, the number of exogenous cells introduced is about 1 to about 25 cells, or about 30-50% of the total number of cells present in the embryo prior to the introduction of the exogenous cells. In some example embodiments, the number of exogenous cells introduced 3 or 4 days after fertilization is about 5 cells. In some example embodiments, the number of exogenous cells introduced 5 days after fertilization is 8 cells. In some example embodiments, the number of exogenous cells introduced 5 days after fertilization is 9 cells. In some example embodiments, the number of exogenous cells introduced 5 days after fertilization is 10 cells. In some example embodiments, the number of exogenous cells introduced 6 days after fertilization is about 10-20 cells.

[0031] In some example embodiments, the self-renewing exogenous cell is an embryonic stem cell, an extended embryonic stem cell, a pluripotent stem cell, an induced pluripotent stem cell, a multipotent stem cell, a totipotent stem cell, a primordial germ cell, a primordial germ cell-like cell, a totipotent cell, or a combination thereof.

[0032] In some example embodiments, the non-human animal embryo is genetically germline ablation. In some example embodiments, the non-human animal embryo comprises any of the preceding paragraphs or one or more engineered cells as described elsewhere herein, or is composed of such cells. In some example embodiments, the self-renewing exogenous cell is germline capable. In some example embodiments, the self-renewing exogenous cell is an engineered cell containing one or more gene modifications in one or more target genes, and wherein said one or more target genes are not NANOS3. In some example embodiments, the self-renewing exogenous cell is not genetically modified. In some example embodiments, the self-renewing exogenous cell comprises an elite genome, a genome-selected genome, or both.

[0033] In some example embodiments, the non-human animal is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, rat, hare, or guinea pig.

[0034] Some example embodiments of this document describe complementary nonhuman embryos produced by any of the preceding paragraphs or by methods of embryo complementation as described elsewhere in this document.

[0035] Some example embodiments describe non-human animals that are produced from the embryos and offspring described in any of the preceding paragraphs.

[0036] These and other aspects, objects, features, and advantages of the exemplary embodiments will become apparent to those skilled in the art in light of the following detailed description of the exemplary embodiments. Attached Figure Description

[0037] The features and advantages of the invention will be understood by referring to the following detailed description of illustrative embodiments, in which the principles of the invention can be utilized, and in the accompanying drawings:

[0038] Figure 1 A schematic diagram of an exemplary alternative male breeding system is shown. Light gray (“host”) represents the steps for producing the host animal. Pathways A and B represent potential alternative sources and steps for producing donor cells. For each donor cell production pathway, the germline complementation step is thus identified. Dark gray represents alternative males used to breed offspring of the donor line.

[0039] Figure 2 A schematic diagram of the CRISPR-Cas target sites within the NANOS3 gene is shown.

[0040] Figure 3 A diagram of bovine NANOS3 exon 1 with the selected dgRNA_4+7 genomic location is shown.

[0041] Figures 4A-4F It demonstrates the production of NANOS3- / - live calves. Figures 4A-4B Images of CRISPR-Cas9 NANOS3-targeted bovine embryos transferred to recipient cows are shown. Figure 4C-4D It shows the result of Figures 4A-4B An image of a one-day-old calf produced from an embryo. Figure 4E An image of a 2-month-old calf is shown. Figure 4F The results of PCR for NANOS3 using DNA from calves are shown. The letters indicate the different alleles present in each animal.

[0042] Figures 5A-5D The results of genotypic analysis of a third live NANOS3 gene-edited calf named Frodo are shown. Figure 5A An image of Frodo at 1 week old is shown. Figure 5B The results of PCR analysis of NANOS3 using DNA obtained from Frodo are shown. Figures 5C1-5C3 A diagram of bovine NANOS3 exon 1 with selected dual gRNA_4+7 genomic locations and Sanger sequencing results showing biallelic, homozygous in-frame mutations (SEQ ID NO:48-51). Figure 5C2 gRNA4 is shown to produce single base pair (bp) substitutions (C to T) and 3bp deletions (SEQ ID NO:48-49). Figure 5C3The gRNA7 that produces a 6bp deletion (SEQ ID NO:50-51) is shown. Figure 5D A comparison is shown between the bovine wild-type NANOS3 exon 1 protein sequence (SEQ ID NO:52) and the Frodo-predicted protein sequence (SEQ ID NO:53). Amino acid substitutions are highlighted in gray and italics (P to L). Three missing amino acids are indicated by * in the wild-type sequence and by dashes in the Frodo sequence. Highly conserved zinc finger binding domains are underlined.

[0043] Figure 6 The structural annotation of sheep NANOS3 exon 1 is shown in dark gray, with key domains in light gray, sgRNA binding sites in dark gray arrows, PAM sites in light gray bars, and primer binding sites in dark gray bars.

[0044] Figure 7 The results of in vitro cleavage assays are shown, demonstrating CRISPR-Cas9 sheep NANOS3 cleavage. Lanes are as follows: Invitrogen 1kb+ sequence ladder labeled "L", sgRNA 1-5, and H2O negative control labeled "-". Genomic DNA amplified by 749bp NANOS3 PCR was minimally cleaved by sgRNA 1, efficiently cleaved by sgRNA 2, 3, and 4, and moderately cleaved by sgRNA 5.

[0045] Figures 8A-8B Images of EGFP-ESC cell lines used to optimize conditions for obtaining embryonic chimeras are shown. Figure 8A The colonies of EGFP-ESC in the culture are shown. Figure 8B The collected and dissociated EGFP-ESCs are shown. Magnified 20x. The second ESC line was derived from female embryos in the early blastocyst stage and cultured in the previously described N2B27 medium. These cells were plated in a different matrix, namely pornectin, without mouse embryonic fibroblast (MEF) feeder cells, with the aim of obtaining a pure bovine cell line.

[0046] Figures 9A-9D The image shows an early stage embryo after injection. Figure 9A A representative bright-field image of a GFP-positive bovine embryo is shown. Figure 9B A representative fluorescence micrograph showing GFP fluorescence (green, expressed in grayscale) is shown. Figure 9C It shows Figures 9A-9B The images in the image are merged. These embryos can be used to derive ESCs for embryonic complementation. In other words, these embryos can be used to derive ESCs for injection into a host embryo. Figure 9DThis shows the injection of embryonic stem cells expressing GFP (such as those derived from...). Figures 9A-9C The embryo in the morula stage (shown as stem cells produced by the embryo).

[0047] Figure 10 Representative immunofluorescence images of ESCs stained with DAPI as a nuclear marker (blue, in grayscale), anti-OCT4 antibody (red, in grayscale), and anti-SOX2 antibody (green, in grayscale) are shown, along with an overlay of the three markers. The images are magnified 20 times.

[0048] Figure 11 The sequence of ESC injections into the embryo using a microinjection system is shown.

[0049] Figure 12A-12D The study shows an ESC incubated in a fluorescent dye and the ESC injected into a host embryo. Figure 12A Images of bovine ESCs after incubation with PKH26 red fluorescent dye and embryos are shown. Magnified 20x, color fluorescence is represented in grayscale. Figure 12B Images of an embryo in the blastocyst stage, injected with PKH26 (red) ESCs, are shown using a confocal cell imaging system. Red fluorescent cells were detected in the ICM. Figure 12C Representative images of embryos at the blastocyst stage after injection of PKH26 (red) ESCs are shown. Embryos were fixed and stained with DAPI (blue, shown in grayscale) to detect cell nuclei. Red fluorescent cells were detected in the ICM. Images are magnified 20x. Figure 12D Representative immunofluorescence images of embryos in the blastocyst stage after injection of PKH26 (red dye) ESCs are shown. Embryos were fixed and stained with DAPI (blue fluorescence, shown in grayscale) to detect cell nuclei and anti-SOX2 antibody (green fluorescence, shown in grayscale) as a marker of pluripotency and ICM. Red fluorescent cells were detected in the ICM, and pluripotency was shown at 20-fold magnification.

[0050] Figures 13A-13D This image shows a representative immunofluorescence image of an embryo on day 8 of development, following an ESC injection on day 6 post-fertilization. Figure 13A DAPI as a nuclear marker, Figure 13B Anti-SOX2 green fluorescent antibody, ( Figure 13C PHK26 and ( Figure 13D Image overlay with coloring. Colored fluorescence is represented in grayscale. Image magnified 20 times.

[0051] Figures 14A-14D This image shows a representative immunofluorescence image of an embryo on day 8 of development, following an ESC injection on day 6 post-fertilization. Figure 14A DAPI as a nuclear marker, Figure 14B Anti-SOX2 green fluorescent antibody, ( Figure 14C PHK26 and ( Figure 14D Image overlay with coloring. Colored fluorescence is represented in grayscale. Image magnified 20 times.

[0052] Figures 15A-15D This image shows a representative immunofluorescence image of an embryo on day 8 of development, following an ESC injection on day 6 post-fertilization. Figure 15A DAPI as a nuclear marker, Figure 15B Anti-SOX2 green fluorescent antibody, ( Figure 15C PHK26 and ( Figure 15D Image overlay with coloring. Colored fluorescence is represented in grayscale. Image magnified 20 times.

[0053] Figure 16 The image shows a plasmid diagram of the FUW plasmid (Addgene plasmid number 14882).

[0054] Figure 17 The combined bright-field and fluorescence microscopy images demonstrate the clonal growth of male Jersey embryonic stem cells transduced with EGFP lentivirus.

[0055] Figures 18A-18B It shows ( Figure 18A NANOS3 PCR was performed on DNA extracted from the tail of a fetus on day 90. (Source: Figure 18B Number 3987 and ( Figure 18C Image of the testes of fetus number 5069 on day 90.

[0056] Figure 19 UMAP maps of different cell populations in the fetal testis are shown. Clusters were identified based on the expression of highly conserved marker genes. PGCs comprised 9% of all cells.

[0057] Figure 20 UMAP plots for individual samples (n=4) are shown.

[0058] Figure 21 The UMAP plot of the processed sample is shown, which shows that only the control (CT) sample is present in the PGC cluster.

[0059] Figures 22A-22F The expression of highly conserved pluripotency, early PGC, and late PGC markers was shown, indicating that most 90d PGCs are in a late stage. Figure 22A -POUF(OCT4), Figure 22B -NANOG、 Figure 22C-NANOS3、 Figure 22D -KIT、 Figure 22E -DAZL、 Figure 22F -DDX4(VASA)).

[0060] Figures 23A-23B A violin expression plot showing late PGC markers was never shown, but the lack of germ cell marker expression in the NANOS3 KO sample compared to the control (CT) sample was never shown.

[0061] Figure 24 The general steps of an in vitro embryo production method using CRISPR-Cas9 to produce NANOS3 KO embryos are shown.

[0062] Figure 25 The sample collection and analysis timeline for scRNA-Seq analysis of gonads from NANOS3 KO animals is shown.

[0063] Figure 26 The NANOS3 KO efficiency using different gRNAs targeting NANOS3 is shown.

[0064] Figures 27A-27B Images of fetal testes from two different NANOS3 KO fetuses are shown.

[0065] Figure 28 The results of PCR detection of NANOS3 in DNA extracted from the blood of a NANOS3 KO fetus are shown.

[0066] Figures 29A-29D -NANOS3 KO bulls are germ-aborted, but otherwise exhibit normal reproductive development. Figure 29A DNA extracted from the blood of bull number 838 (“Fauci”) was subjected to NANOS3 PCR. The letters AD indicate different alleles. Number 838 is a mosaic KO with 4+ alleles, including one large deletion, and no wild type. Figure 29B The 1-day-old NANOS3 KO Bulls, number 838. Figure 29C )12-month-old NANOS3 KO bull, number 838. Figure 29D Results of the 12-month Breeding Steadyness Examination (BSE) for NANOS3 KO bull number 838.

[0067] Figures 30A-30C Physiological characteristics of NANOS3 KO bull number 838. Figure 30A Image of bull number 838, who is 15 months old. Figure 30B Image of the reproductive tract of bull number 838. Figure 30CRepresentative images of H&E-stained cross-sections of testes from age-matched wild-type (NANOS3+ / +) bulls (left panel) compared to bull number 838 (NANOS3- / -) (right panel). Both samples possessed Sertoli cells lining the seminiferous tubules, but bull number 838 lacked any spermatogenesis.

[0068] Figures 31A-31B - Physiological characteristics of bull number 3964. Figure 31A Image of a 15-month-old bull, number 3964. Figure 31B Image of the reproductive tract of bull number 3964.

[0069] Figures 32A-32F Physiological characteristics of heifer number 854 (NANOS3 KO). Figure 32A Image of a 15-month-old heifer, number 854. Figure 32B Image of the reproductive tract of heifer number 854. Figure 32C-32D The left ovary of heifer number 854 Figure 32C ) and the presumed original bar on the right ( Figure 32D (Image of ). Figure 32E-32F The images show representative cross-sections of an ovary stained with H&E staining in a completely oocyte-deficient state at different magnifications. Figure 32E From Figure 32C The ovary shown. Figure 32F From Figure 32D The ovary shown.

[0070] Figure 33 PCR of -DDX3 was used for fetal sex determination. A single band indicates female, and two bands at 184 and 208 bp indicate male. Cosmo the bull was used as a positive control.

[0071] Figure 34 -PCR was used to detect GPF. The absence of a band at 425 bp indicates the absence of GFP in the fetal sample. Cosmo DNA was used as a positive control.

[0072] Figure 35 Genotypic analysis of CRISPR / CAS9 NANOS3-targeted bovine samples. NANOS3 remote PCR results. The wild-type (+) band was 6,274 bp in size. Bands smaller than the wild-type band indicated large (>500 bp) deletions in NANOS3.

[0073] Figure 36- An overview of the pluripotency states of mouse, bovine, porcine, and equine stem cells derived under different culture conditions. mESC: mouse embryonic stem cells (primordial state). FTW-mESC: morphological mouse embryonic stem cells, Yu et al., (2021). EpiSC: ectoderm stem cells (primordial state). bEPSC: bovine extended potential stem cells (Zhao et al., 2021). bESC: bovine embryonic stem cells (Bogliotti et al., 2018, Proceedings of the National Academy of Sciences, 115, 2090-2095). pEPSC: porcine extended potential stem cells (Zhao et al., 2021, Proceedings of the National Academy of Sciences, 118, 9). FTW-equi: morphological equine stem cells (Yu et al., Cell Stem Cell, 28, 550-567, (2021)).

[0074] Figure 37 - A representative image of a hypothetical NANOS / knockout embryo, supplemented with red (as shown in gray) bEPSC stained with DAPI (blue, as shown in gray) and anti-SOX2 antibody (green, as shown in gray) as nuclear markers. Overlay of the three channels. Magnified 20x.

[0075] Figure 38 - A representative day 7 blastocyst with red-stained (e.g., gray-scale) ESCs on the day of embryo transfer.

[0076] Figure 39 - Representative images of two recovered embryos under a stereomicroscope, with arrows pointing to the blastodisc.

[0077] Figures 40A-40B - qPCR was performed on ESCs carrying the EF1a-tdTomato marker during the first DNA extraction from the elongated embryonic placental tissue.

[0078] Figures 41A-41B - During the second DNA extraction from the elongated embryonic placental tissue, qPCR was performed to detect ESCs carrying the EF1a-tdTomato marker.

[0079] Figure 42 - A representative immunofluorescence image of tdTomato-expressing cells stained with anti-TdTomato protein (green, if presented in grayscale). Magnified 20x.

[0080] Figure 43- A representative image of the ESC cells in the ICM of the blastocyst after 5-cell ESCs were injected into a day 5 morula. There is clear evidence that these 5 ESCs expanded in the ICM of the expanding blastocyst, as shown by red fluorescence (indicated in grayscale and indicated by black arrows).

[0081] Figure 44 - Representative images of embryos collected during the blastocyst stage and transferred to the recipient. Magnified 20x.

[0082] Figure 45 -qPCR assays were performed on ESCs carrying the EF1a-tdTomato marker in DNA extracted from placental tissues derived from three elongated embryos injected with ESCs expressing tdTomato (from recipients 1076 and 1125) and seven embryos injected with ESCs carrying a green fluorescent marker (from recipients 1074 and 1078).

[0083] The accompanying figures in this article are for illustrative purposes and are not necessarily drawn to scale. Detailed Implementation

[0084] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described, and therefore variations are certainly possible. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred methods and materials are described hereafter.

[0086] All publications and patents referenced in this specification are for the purpose of disclosing and describing methods and / or materials in relation to the referenced publications. All such publications and patents are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is clearly limited to the methods and / or materials described in the referenced publications and patents and does not extend to any dictionary definitions of the referenced publications and patents. Any dictionary definitions in the referenced publications and patents that are not expressly repeated in this application should not be construed as such, nor should they be interpreted as defining any terms appearing in the appended claims. References to any publication are for its publication prior to the filing date and should not be construed as an admission that this disclosure is not entitled to precedence over such publications by virtue of prior disclosure. Furthermore, the publication date provided may differ from the actual publication date, which may require separate confirmation.

[0087] As will be apparent to those skilled in the art upon reading this disclosure, each embodiment in the individual examples described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any embodiment in several other embodiments without departing from the scope or spirit of this disclosure. Any described method may be performed in the order of the described events or in any other logically possible order.

[0088] In the case of indicating a range, another aspect includes from one particular value and / or to another particular value. When a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range (one-tenth of a unit to the lower limit, unless otherwise explicitly stated) and any other stated or intermediate values ​​within the range are covered within this disclosure. The upper and lower limits of these smaller ranges may be independently included in even smaller ranges and are also covered within this disclosure, subject to any explicitly excluded limits within the stated range. Where a stated range includes one or both limits, the range excluding any one or both of the included limits is also included within this disclosure. For example, where a stated range includes one or both limits, the range excluding any one or both of the included limits is also included within this disclosure; for example, the phrase "x to y" includes a range from 'x' to 'y', as well as ranges greater than 'x' and less than 'y'. A range can also be expressed as an upper limit, such as 'about x, y, z or less', and should be interpreted as including the specific range of 'about x', 'about y', and 'about z', as well as the range of 'less than x', 'less than y', and 'less than z'. Similarly, the phrase 'about x, y, z or greater' should be interpreted as including the specific range of 'about x', 'about y', and 'about z', as well as the range of 'greater than x', 'greater than y', and 'greater than z'. Additionally, the phrase 'about 'x' to 'y' includes 'about 'x' to 'y', where 'x' and 'y' are numerical values.

[0089] It should be understood that ratios, concentrations, amounts, and other numerical data may be expressed in range format herein. It should be further understood that each endpoint of a range is significant relative to and independent of the other endpoint. It should also be understood that numerous values ​​are disclosed herein, and in addition to the value itself, each value is disclosed as “about” the specific value. For example, if the value “10” is disclosed, then “about 10” is also disclosed. In this document, a range may be expressed as from “about” one specific value and / or to “about” another specific value. Similarly, when a value is expressed as an approximation using the antecedent “about”, it should be understood that the specific value forms another aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0090] It should be understood that this range format is used for convenience and brevity, and therefore should be interpreted flexibly to include not only the numerical values ​​explicitly stated as the limits of the range, but also all individual numerical values ​​or subranges covering the range, as if each numerical value and subrange were explicitly stated. For example, the numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly stated values ​​of about 0.1% to about 5%, but also the individual values ​​(e.g., about 1%, about 2%, about 3%, and about 4%) and subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2% and about 0.5% to about 4.4%, and other possible subranges) within the indicated range. General definition

[0091] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of commonly used terms and techniques in molecular biology can be found in: *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); *Molecular Cloning: A Laboratory Manual*, 4th edition (2012) (Green and Sambrook); *Current Protocols in Molecular Biology* (1987) (edited by F.M. Ausubel et al.); *Methods in Enzymology* (Academic Press, Inc.); *PCR 2: A Practical Approach* (1995) (edited by M.J. MacPherson, B.D. Hames, and G.G. Taylor); *Antibodies: A Laboratory Manual* (1988) (edited by Harlow and Lane); *Antibodies: A Laboratory Manual*, 2nd edition, 2013 (edited by E.A. Greenfield); *Animal Cell Culture*. Culture (1987) (edited by RIFreshney); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (editors), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (editor), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc.Published by [Publisher Name], 1995 (ISBN 9780471185710); Singleton et al., *Dictionary of Microbiology and Molecular Biology*, 2nd ed., J. Wiley & Sons (New York, NY, 1994); March, *Advanced Organic Chemistry Reactions, Mechanisms and Structure*, 4th ed., J. Wiley & Sons (New York, NY, 1992); Marten H. Hofker and Janvan Deursen, *Transgenic Mouse Methods and Protocols*, 2nd ed. (2011); and Primrose and Twyman, *Principles of Gene Manipulation and Genomics*, 2006, published by Blackwell Publishers.

[0092] Definitions of common terms and techniques in chemistry and organic chemistry can be found in Smith, *Organic Synthesis*, Academic Press, 2016; Tinoco et al., *Physical Chemistry*, 5th edition (2013), Pearson; Brown et al., *Chemistry, The Central Science*, 14th edition (2017), Pearson; Clayden et al., *Organic Chemistry*, 2nd edition, 2012, Oxford University Press; Carey and Sunberg, *Advanced Organic Chemistry, Part A: Structure and Mechanisms*, 5th edition, 2008, Springer; and Carey and Sunberg, *Advanced Organic Chemistry, Part B: Reactions and Synthesis*. Chemistry, Part B: Reactions and Synthesis, 5th edition, 2010, published by Springer, and Vollhardt and Schore, Organic Chemistry, Structure and Function, 8th edition (2018), published by WH Freeman.

[0093] Definitions of commonly used terms, analyses, and techniques in genetics can be found, for example, in Hartl and Clark, *Principles of Population Genetics*, 4th edition, 2006, published by Oxford University Press; *Genetics: Analysis and Principles*, 7th edition, 2021, published by McGraw Hill; Isik et al., *Genetic Data Analysis for Plant and Animal Breeding*, 1st edition, 2017, published by Springer International Publishing AG; Green, EL, *Genetics and Probability in Animal Breeding Experiments*, 2014, published by Palgrave; and Bourdon, RM, *Understanding Animal Breeding*. 2nd edition, 2000, by Prentice Hall; Pal and Chakravarty, *Genetics and Breeding for Disease Resistance of Livestock*, 1st edition, 2019, by Academic Press; Fasso, D., *Classification of Genetic Variance in Animals*, 1st edition, 2015, by Callisto Reference; Megahed, M., *Handbook of Animal Breeding and Genetics*, 2013, by Omniscriptum GmbH & Co. Kg., LAP Lambert Academic Publishing; Reece, *Analysis of Genes and Genomes*.2004, published by John Willie & Sons; Deonier et al., *Computational Genome Analysis*, 5th edition, 2005, published by Springer-Verlag, New York; Meneely, P., *Genetic Analysis: Genes, Genomes, and Networks in Eukaryotes*, 3rd edition, 2020, published by Oxford University Press.

[0094] As used herein, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include both the singular and plural referents.

[0095] As used herein, when used in conjunction with measurable variables such as parameters, quantities, durations, etc., the terms “about,” “approximately,” or “substantially” mean to cover a specified value or variation from a specified value, including variations within experimental error (which can be determined, for example, by a given dataset, prior art accepted standards, and / or, for example, by a given confidence interval (e.g., a confidence interval of 90%, 95%, or greater than the mean), such as a variation of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of a particular value), provided that such variation is suitable for implementation in the disclosed invention. As used herein, the terms “about,” “approximate,” “equal to or about,” and “substantially” can mean that the quantity or value in question can be an exact value or a value that provides an equivalent result or effect to the result or effect pursuant to the claims or taught herein. In other words, it should be understood that quantities, sizes, formulations, parameters, and other quantities and characteristics are not and need not be precise, but may be approximate and / or larger or smaller as needed to reflect tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art, resulting in equivalent results or effects. In some cases, it is not reasonable to determine a value that provides an equivalent result or effect. Generally, quantities, sizes, formulations, parameters, or other quantities or characteristics are “about,” “approximately,” or “equal to or about,” whether explicitly stated or not. It should be understood that where “about,” “approximately,” or “equal to or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless otherwise specifically stated.

[0096] The terms “optional” or “optionally” mean that the event, situation or substituent described below may or may not occur, and the description includes instances where the event or situation occurs and instances where the event or situation does not occur.

[0097] The enumeration of the range of numbers by endpoints includes all numbers and fractions that fall into the corresponding range, as well as the listed endpoints.

[0098] As used herein, a “biological sample” means a sample obtained from, prepared by, secreted by, excreted by, or otherwise containing a part of or derived from a living organism (e.g., an individual). Biological samples may contain whole cells and / or live cells and / or cell debris, and / or cell products, and / or viral particles. Biological samples may contain (or be derived from) “body fluids.” Biological samples may be obtained from the environment (e.g., water sources, soil, air, etc.). Such samples are also referred to herein as environmental samples. As used herein, “body fluid” means any non-solid excretion, secretion, or other fluid present in a living organism, and includes, but is not limited to, amniotic fluid, aqueous humor, vitreous fluid, bile, blood or components thereof (e.g., plasma, serum, etc.), breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and mucus), pericardial fluid, peritoneal fluid, pleural fluid, pus, nasal discharge, saliva, semen (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and one or more mixtures thereof. Biological samples include cell cultures, body fluids, and cell cultures derived from body fluids. Body fluids can be obtained from a living organism, for example, by puncture or other collection or sampling procedures.

[0099] As used herein, “blastocyst” refers to an early stage of embryonic development, which comprises the inner cell mass (from which the embryonic body arises) and a fluid-filled cavity typically surrounded by a single layer of trophoblast cells. (Developmental Biology, 6th Edition, edited by Scott F. Gilbert, Sinauer Associates, Inc., Publishers, Sunderland, Mass. (2000))

[0100] As used herein, the terms “encoding” or “coded” refer, with respect to a specific nucleic acid, to information transcribed into RNA, and in some cases, to information translated into a specific protein. Nucleic acids encoding proteins may contain insert sequences (e.g., introns) within the translational region of the nucleic acid, or may lack such intermediate untranslated sequences (e.g., as in cDNA). The information encoding proteins is specified by the use of codons. Typically, amino acid sequences are encoded by nucleic acids using a “universal” genetic code. When nucleic acids are synthesized or altered, the known codon preferences of the intended host for the nucleic acid to be expressed can be utilized. As used herein, regarding relationships between DNA, cDNA, cRNA, RNA, proteins / peptides, etc., “corresponding” or “encoding” (which may be used interchangeably herein) refers to a potential biological relationship between these different molecules. Therefore, those skilled in the art will understand that, given the sequence of any other molecule having a similar biological relationship to these molecules, operably “corresponding” can guide the determination of possible potential and / or resulting sequences of other molecules. For example, an RNA sequence can be determined from a DNA sequence, and a cDNA sequence can be determined from an RNA sequence.

[0101] As used herein, “heterologous” in relation to nucleic acids refers to nucleic acids derived from a foreign species, or, if derived from the same species, nucleic acids that have been substantially modified from their natural form at a composition and / or genomic locus through intentional human intervention. For example, a promoter operatively linked to a heterologous structural gene originates from a species different from the source of said structural gene, or, if derived from the same species, one or both promoters have been substantially modified from their original form. Heterologous proteins can originate from a foreign species, or, if derived from the same species, have been substantially modified from their original form through human intervention.

[0102] As used herein, the term "early stage embryo" refers to any embryonic stage between a fertilized egg and a blastocyst. Typically, embryos at the eight-cell stage and the morula stage are referred to as early stage embryos.

[0103] As used herein, “embryonic stem cells” or “ES cells” refers to cultured cells derived from the inner cell mass of an early-stage embryo that can be genetically modified to maintain their pluripotency and, if injected into a host embryo, can contribute to all organs of the resulting chimeric animal. Developmental Biology, 6th Edition, edited by Scott F. Gilbert, Singall Associates, Sunderland, MA (2000).

[0104] As used herein, “primordial germ cells” means those cells that are produced in the early stages of embryonic development, which produce the spermatogenic lineage through the intermediate stage of germ cells or the female lineage through the intermediate stage of oogonia.

[0105] As used herein, “self-renewal” refers to the ability of an undifferentiated cell to divide while maintaining an undifferentiated state in at least one of its progeny cells, thereby maintaining or expanding a population of undifferentiated cells, while optionally generating differentiated cells or cell populations. Therefore, the term “self-renewing cell” as used herein is an undifferentiated cell with the ability to divide and optionally differentiate, in which case, at least one of its progeny cells remains undifferentiated, thereby allowing for the maintenance or expansion of a population of undifferentiated cells.

[0106] As used herein, "pluripotent" refers to the ability of a cell to differentiate into any of the three germ layers (endoderm, mesoderm, and ectoderm). Therefore, as used herein, the term "pluripotent cell" is a cell capable of differentiating into or producing any of the three germ layers. Thus, a pluripotent cell has the ability to divide into most of the cells of an organism, but cannot develop independently into a complete organism.

[0107] As used herein, "totipotent" refers to the ability of a cell or population of cells to differentiate into any cell type (e.g., blastomeres) or a complete embryo or animal (including the placenta). Therefore, the term "totipotent cell," as used herein, is a cell that has the ability to differentiate into or produce any cell type (e.g., blastomeres) or a complete embryo or animal (including the placenta). In other words, a totipotent cell can develop itself into a complete organism. For example, a fertilized egg is totipotent. Totipotent cells have the ability to divide until they form an entire embryo or animal.

[0108] As used herein, “selected genome” or “selected genotype” refers to cells, tissues, animals, etc., selected based on one or more DNA sequences of their genome. The techniques used to determine the genome sequence and genotype of any particular locus are generally known in the art and include all molecular biology methods of genomic and DNA analysis, population genetics-based methods based on genetic principles, and combinations thereof.

[0109] As used in this article, "fertilized egg" refers to a single-cell embryo.

[0110] As used herein, the terms “recombinant” or “engineered” generally refer to non-naturally occurring nucleic acids, nucleic acid constructs, or peptides. Such non-naturally occurring nucleic acids can include modified native nucleic acids, such as native nucleic acids with deletions, substitutions, inversions, insertions, etc., and / or combinations of nucleic acid sequences from different sources conjugated using molecular biology techniques (e.g., nucleic acid sequences encoding fusion proteins (e.g., proteins or peptides formed by a combination of two different proteins or protein fragments)), combinations of nucleic acids encoding peptides with promoter sequences, wherein the coding and promoter sequences originate from different sources or would not typically occur together naturally (e.g., nucleic acids and constitutive promoters), etc. Recombinant or engineered can also refer to peptides encoded by recombinant nucleic acids. Non-naturally occurring nucleic acids or peptides include nucleic acids and peptides modified by humans.

[0111] As used herein, the term "allergen" refers to an antigen, microorganism, plant, or product thereof that elicits an abnormal immune response, in which the immune system repels a perceived threat that would otherwise be harmless to the body. Allergens can be found in a wide variety of sources (e.g., animal products (e.g., meat, milk, and products made from them), food, insects, mold spores, plants, and chemicals). Allergens can include, but are not limited to, dust mites, pollen, spores, poison ivy, poison oak, pet dander, royal jelly, peanuts (legumes), nuts, insect bites, seafood, and shellfish.

[0112] As used herein, “culture” can refer to maintaining cells under conditions in which they can proliferate as a group of cells and avoid senescence. “Cultivation” can also include conditions in which cells also differentiate or alternatively differentiate. Culture may include one or more steps or conditions, and one or more steps may include passage, cell transfer, change of culture medium, change of culture temperature, change of atmospheric gas, etc.

[0113] As used herein, the terms “nucleic acid,” “nucleotide sequence,” and “polynucleotide” are used interchangeably and generally refer to a string of at least two base-sugar-phosphate combinations, and specifically to single-stranded and double-stranded DNA, DNA of mixtures of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA of mixtures of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA, which may be single-stranded, or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Additionally, as used herein, a polynucleotide may refer to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The strands in such regions may originate from the same molecule or different molecules. These regions may include all of one or more molecules in a molecule, but more typically only regions of some molecules in a molecule. One molecule in a triple-stranded region is typically an oligonucleotide. “Polynucleotide” and “nucleic acid” also encompass such chemically modified, enzymatically modified, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA specific to viruses and cells (especially simple and complex cells). For example, the term polynucleotide as used herein can include DNA or RNA containing one or more modified bases as described herein. Thus, to name just two examples, DNA or RNA containing anomalous bases (such as inosine) or modified bases (such as triphenylmethylated bases) is a polynucleotide as used herein. "Polynucleotide," "nucleotide sequence," and "nucleic acid" also include other variants of the phosphate backbone of PNA (peptide nucleic acid), phosphate thioesters, and native nucleic acids. Native nucleic acids have a phosphate backbone, while artificial nucleic acids may contain other types of backbones but contain the same bases. Therefore, DNA or RNA with a modified backbone is a "nucleic acid" or "polynucleotide" as the term implies, for stability or other reasons. As used herein, "nucleic acid sequence" and "oligonucleotide" also encompass nucleic acids and polynucleotides as defined elsewhere herein.

[0114] As used herein, the term "fragment" referring to a peptide, polypeptide, or protein, as used throughout this specification, generally means a portion of the peptide, polypeptide, or protein, such as a truncated N-terminal and / or C-terminal form of the peptide, polypeptide, or protein. Preferably, the fragment may contain at least about 30% of the amino acid sequence length of the peptide, polypeptide, or protein, for example, at least about 50% or at least about 70%, preferably at least about 80%, for example, at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% or even about 99%. For example, within a length not exceeding that of a full-length peptide, polypeptide, or protein, a fragment may comprise a sequence of ≥5, ≥10, ≥20, or ≥30 consecutive amino acids, for example, ≥40, ≥50, ≥60, ≥70, ≥80, ≥90, ≥100, ≥200, ≥300, ≥400, ≥500, ≥500, or ≥600 consecutive amino acids, corresponding to a full-length peptide, polypeptide, or protein. The term "fragment" in relation to nucleic acids (polynucleotides) generally refers to a 5' truncated and / or 3' truncated form of the nucleic acid. Preferably, the fragment may contain at least about 30% of the nucleic acid sequence length of the nucleic acid, for example, at least about 50% or at least about 70%, preferably at least about 80%, for example, at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% or even about 99%. For example, within the range not exceeding the length of a full-length nucleic acid, a fragment may include a sequence of ≥5, ≥10, ≥20, or ≥30 consecutive nucleotides corresponding to the full-length nucleic acid, such as ≥40, ≥50, ≥60, ≥70, ≥80, ≥90, ≥100, ≥200, ≥300, ≥400, ≥500, or ≥600 consecutive nucleotides. The terminology encompasses fragments produced by any mechanism, in vivo and / or in vitro, such as, but not limited to, selective transcription or translation, external and / or internal proteolysis, external and / or nuclear dissolution, or degradation of peptides, polypeptides, proteins, or nucleic acids, such as by physical, chemical, and / or enzymatic proteolysis or nuclear dissolution.

[0115] As used herein, “expression” refers to the process by which polynucleotides are transcribed into RNA transcripts. In the context of mRNA and other translated RNA species, “expression” also refers to the process by which transcribed RNA is subsequently translated into peptides, polypeptides, or proteins. In some cases, “expression” can also reflect the stability of a given RNA. For example, when measuring RNA, an increase / decrease in RNA transcription levels may be a result of increased / decreased transcription and / or increased / decreased RNA transcriptional stability and / or degradation, depending on the methods used to detect and / or quantify RNA and other techniques used in conjunction with RNA detection and / or quantification. Those skilled in the art will understand the relationship between these techniques and “expression” in these different contexts and underlying biological mechanisms.

[0116] As used herein, “reduced expression” or “low expression” means a decrease or reduction in the expression of a gene (such as a gene associated with an antigen processing pathway) or its gene product in a sample compared to the expression of the gene or gene product described in a suitable control. As used throughout this specification, “suitable control” is a control included that will be readily understood by one of ordinary skill in the art, enabling the determination of whether the variable being evaluated is an effect, such as a desired or hypothesized effect. One such control will also be readily understood by one of ordinary skill in the art, particularly based on context, the variable, the desired or hypothesized effect, and what constitutes a suitable or appropriate control. In one embodiment, the control is a sample from a healthy individual or other normal individual. By way of non-limiting example, if the sample is a lung tumor sample and contains lung tissue, the control is lung tissue from a healthy individual. The term “reduced expression” preferably means a reduction of at least 25% relative to such a control, for example, a reduction of at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

[0117] The term "modification leading to the reduced expression" refers to a modification of a gene that affects the expression level of the gene or another gene, resulting in a decrease or reduction in the expression level of the gene or another gene. In a particular embodiment, the modification occurs in a gene associated with an antigen processing pathway. In some embodiments, the modification occurs in a gene associated with a cross-presentation pathway. The modification can be any nucleic acid modification, including but not limited to mutation, deletion, insertion, substitution, ligation, digestion, breakage, and frameshift. The modification is preferably selected from the group consisting of free mutation, deletion, and frameshift. In a particular embodiment, the modification is a mutation that leads to a decrease in the expression of a functional gene product.

[0118] As used herein, both “increased expression” and “overexpression” refer to an increase in the expression of a gene (such as a gene associated with antigen treatment and / or presentation pathway) or its gene product in a sample compared to the expression of the gene or gene product described in a suitable control. The term "increased expression" preferably refers to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, or 3% relative to a suitable control. 90%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 80 0%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000%, 1010%, 1020%, 1030%, 1040%, 1050%, 1060%, 1070%, 1080%, 1090%, 1100%, 1110%, 1120%, 1130%, 1140%, 1150%, 1160%, 1170%, Expressions of 1180%, 1190%, 1200%, 1210%, 1220%, 1230%, 1240%, 1250%, 1260%, 1270%, 1280%, 1290%, 1300%, 1310%, 1320%, 1330%, 1340%, 1350%, 1360%, 1370%, 1380%, 1390%, 1400%, 1410%, 1420%, 1430%, 1440%, 1450%, 1460%, 1470%, 1480%, 1490% or up to 1500% or more.

[0119] The term "modification leading to the increased expression" refers to a modification of a gene that affects the expression level of the gene or another gene, resulting in an increase in the expression of the gene or another gene. In a particular embodiment, the modification occurs in a gene associated with an antigen processing pathway. In some embodiments, the modification occurs in a gene associated with a cross-presentation pathway. The modification can be any nucleic acid modification, including but not limited to mutation, deletion, insertion, substitution, ligation, digestion, breakage, and frameshift. The modification is preferably selected from the group consisting of free mutation, deletion, and frameshift. In a particular embodiment, the modification is a mutation that leads to a decrease in the expression of a functional gene product.

[0120] As used herein, "gene" can refer to a genetic unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and contains the genetic instructions for a characteristic or trait in an organism. The term "gene" can refer to the translated and / or untranslated region of the genome. "Gene" can also refer to a specific sequence of DNA transcribed into RNA transcripts, which can be translated into polypeptides or catalytic RNA molecules, including but not limited to tRNA, siRNA, piRNA, miRNA, long non-coding RNA, and shRNA.

[0121] As used herein, “gene product” refers to any polynucleotide transcribed (in vivo or in vitro) into an RNA molecule. The term “gene product” also refers to a polypeptide translated from an RNA gene product.

[0122] As used herein, “polypeptide” or “protein” refers to a sequence of amino acid residues. These sequences are written from left to right, from the amino group to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are named with three-letter or single-letter codes, as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). “Protein” and “polypeptide” can also refer to molecules composed of one or more chains of amino acids arranged in a specific sequence. The term "protein" is used interchangeably with "polypeptide." The sequence is determined by the base sequence of nucleotides in the gene that encodes the protein. Proteins are necessary for the structure, function, and regulation of the body's cells, tissues, and organs.

[0123] As used herein, “cell population” refers to any number of cells greater than 1, but preferably at least 1 x 102 3 1 cell, at least 1 x 10 4 1 x 10 cells, at least 1 x 10 5 1 cell, at least 1 x 10 6 1 cell, at least 1 x 10 7 1 cell, at least 1 x 10 8 1 cell, at least 1 x 10 9 One cell or at least 1 x 10 10 Each cell.

[0124] As used herein, the term "molecular weight" generally refers to the mass or average mass of a material. For polymers or oligomers, molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized by various methods, including gel permeation chromatography (GPC) or capillary viscosity analysis. GPC molecular weight is reported as a number-average molecular weight (M0.05). n Conversely, weight-average molecular weight (M) w Capillary viscosity analysis provides an estimate of molecular weight as the intrinsic viscosity determined by diluting a polymer solution using a specific set of concentration, temperature, and solvent conditions.

[0125] As used herein, a “targeting moiety” refers to a molecule, complex, agent, etc., capable of specifically or selectively interacting, binding, or acting with a target molecule, agent, and / or complex, which is associated with, is part of, or is coupled to another object, complex, surface, etc. (e.g., cell or cell population, tissue, organ, subcellular site, object surface, particle, etc.). The targeting moiety can be chemical, biological, metallic, polymeric, or other agents and molecules with targeting capabilities. The targeting moiety can be an amino acid, peptide, polypeptide, nucleic acid, polynucleotide, lipid, sugar, metal, small molecule chemical, or a combination thereof. The targeting moiety can be an antibody or fragment thereof, aptamer, DNA, RNA (e.g., guide RNA for RNA-guided nucleases or systems), ligand, substrate, enzyme, or a combination thereof. The specificity or selectivity of the targeting moiety can be determined by any suitable method or technique understood by one of ordinary skill in the art. For example, in some embodiments, the methods described herein include determining the dissociation constants of the targeting moiety and the target. In some embodiments, the targeting portion is specific, and under the conditions employed, such as intracellular or physiological conditions consistent with cell survival, the equilibrium dissociation constant Kd is 10. -3 M or smaller, 10 -4 M or smaller, 10 -5 M or smaller, 10 -6 M or smaller, 10-7 M or smaller, 10 -8 M or smaller, 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller or 10 -12 M or less. In some embodiments, specific binding can be achieved through multiple weaker interactions (e.g., multiple individual interactions, where each individual interaction is characterized by a value greater than 10). -3 This is achieved through (M = Kd). In some embodiments, the targeting portion has increased binding, association, interaction, and activity, such as an increase of 1 to 500 times or more, compared to the non-target portion. The target of the targeting portion can be amino acids, peptides, polypeptides, nucleic acids, polynucleotides, lipids, sugars, metals, small molecule chemicals, combinations thereof, etc. The target can be receptors, biomarkers, transport proteins, antigens, complexes, combinations thereof, etc.

[0126] The terms “subject” and “individual” are used interchangeably herein to refer to vertebrates, preferably mammals, more preferably cattle. Mammals include, but are not limited to, rats, monkeys, humans, farm animals, racing animals, and pets. Such terms also cover tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0127] As used in this article, “wild type” is the average form of an organism, variant, strain, gene, protein, or trait that appears in a given population in nature, and is different from the mutant forms that may be produced by selective breeding, recombinant engineering, and / or transgenic transformation.

[0128] Various embodiments are described below. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation on the broader aspects discussed herein. An aspect described in connection with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment. Throughout this specification, the references to “an embodiment,” “an example embodiment,” or “example embodiment” mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Therefore, the appearance of the phrases “in one embodiment,” “in one embodiment,” or “example embodiment” throughout this specification does not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, in one or more embodiments, specific features, structures, or characteristics can be combined in any suitable manner as will be apparent to those skilled in the art based on this disclosure. Moreover, while some embodiments described herein include some but not all features included in other embodiments, combinations of features from different embodiments are intended to be within the scope of the invention. For example, in the appended claims, any claimed embodiment can be used in any combination.

[0129] All publications, published patent documents and patent applications cited herein are hereby incorporated by reference to the extent that each individual publication, published patent document or patent application is specifically and individually indicated as being incorporated by reference. Overview

[0130] Conventional genetic selection and breeding programs have generated elite genetic seed reserves. However, there is a lag in genetic improvement between these elite nuclear seed reserves and commercial animals. This lag is due to the generational intervals in conventional breeding programs. Therefore, methods are needed to increase the rate of genetic improvement, particularly at the commercial animal level.

[0131] Germ complementation, which involves the use of germline defective hosts, is a method that can be used to effectively disseminate animals with superior genetics and / or traits. One method for generating germline defective hosts is through treatment with chemically toxic agents (e.g., busulfan) or local irradiation; however, these methods are not effective in livestock because they either fail to completely eliminate endogenous germlines or have undesirable side effects on animal health. A promising alternative is the use of GnEd to knock out genes (such as NANOS2 or DAZL) essential for the production of the animal's own germ cells in fertilized eggs. NANOS2 is primarily expressed in male germ cells and is essential for maintaining a spermatogonial stem cell population.

[0132] The NANOS gene family, including NANOS3, is essential for germ cell development, although the regulatory processes differ among species and homologs. NANOS3 is present in migrating sexual primordial germ cells (PGCs), and homozygous deficiency of NANOS3 results in the complete loss of male and female germ cells in mice (see, for example, Tsuda et al., Science. 2003. 301(5637):1239-1241). Recently, female NANOS3 knockout embryos were generated using a somatic cell nuclear transfer strategy, and fetal ovaries containing germ cells were observed (Ideta et al., 2016. Scientific Reports. 6:24983). However, there are no reports of knocking out NANOS3 in male cattle or germ-depleted male cattle, or using gene-editing methods to knock out NANOS3 in cattle.

[0133] In other words, the embodiments disclosed herein can provide methods and compositions for germline complementation and are used for germline complementation strategies, such as NANOS3-deficient cells and / or non-human animals, and more specifically, non-human animals undergoing genetic germline ablation (such as cattle). Other compositions, compounds, methods, features, and advantages of this disclosure will be apparent or become apparent to those skilled in the art upon examination of the following figures, detailed descriptions, and examples. All such additional compositions, compounds, methods, features, and advantages are intended to be included within the scope of this specification and disclosure. phylogenetic complementarity

[0134] Several exemplary embodiments described herein illustrate methods for germline complementation utilizing NANOS3-deficient host animals and / or cells, along with suitable donor cells. Host and donor cells and / or animals used in germline complementation methods are described in more detail elsewhere in this document. Figure 1 A general strategy for germline complementation to produce alternative male animals is illustrated. Although not shown, similar methods and strategies can be used to replace female animals. Typically, NANOS3-deficient host animals / embryos lack germ cells (also referred to herein as germline-ablated, genetically germline-ablated, germline-deficient, or germline-deficient) or have the ability to produce germ cells. Germline complementation with suitable donor cells is then used to supplement the host cells with germ cells from the donor cells / animal (or the ability to produce missing germ cells). This can produce alternative male and / or female animals that can be used for natural mating or conventional AI breeding to produce commercial offspring with donor cell genetics.

[0135] During host embryonic development, cell complementarity occurs by injecting donor cells (exogenous cells of the host) into the gonads of the host animal or into the preimplantation host embryo (see, for example, Figure 1In some embodiments, the method of nonhuman animal embryo complementation includes, optionally, introducing a self-renewing exogenous cell population into a nonhuman animal preimplantation embryo at approximately day 3, 4, 5, or 6 post-fertilization; optionally washing the nonhuman animal preimplantation embryo in HEPES or other suitable buffer; and culturing the nonhuman preimplantation embryo in a suitable culture medium, optionally consisting of a suitable bovine medium in a 1:1 volume ratio, such as a medium supplemented with at least N2, B27, FGF, and IWR-1. Suitable embryo culture media may include one or more salts (e.g., sodium chloride, potassium chloride, calcium chloride, potassium dihydrogen phosphate, magnesium sulfate), one or more buffers (e.g., sodium bicarbonate), one or more energy substrates (e.g., glucose, sodium lactate and / or sodium pyruvate), non-essential amino acids or mixtures (e.g., NEAA 8, NEAA 9), one or more glutamine dipeptides (e.g., alanyl-glutamine), one or more essential amino acids or mixtures (e.g., EAA 2, EAA 11, etc.), one or more chelating agents (e.g., EDTA), one or more macromolecules (e.g., hyaluronic acid, HAS, etc.), one or more fatty acids (e.g., lipoic acid, etc.), one or more vitamins (e.g., A, E, D, C, K, B, folic acid), one or more antibiotics and / or antifungal agents, or any combination thereof. Other exemplary embryo culture media include, but are not limited to, M2 medium, cleavage K-SCIM medium, blastocyst K-SIBM medium, Quinns dominant cleavage medium, Quinns dominant blastocyst medium, FERTICUK IVF medium, FERTICULT G3 medium, IVC-TWO medium, IVC-THREE medium, ECM medium, MultiBlast medium, embryo-assisted medium, Blast-assisted medium, ISM1 medium, ISM2 medium, G-1PLUS medium, G-2PLUS medium, IVF medium, CCM medium, BO-IVF medium (ivfbioscience), for example, Thompson and Peterson. 2000. Human Reproduction, Dec; 15 Supplement 5:59-67. doi:10.1093 / humrep / 15.suppl_5.59; Santana et al., Molecular Reproduction and Development (MolReprod. The culture medium described in the following journals: Dev., October 2014; 81(10):918-27. doi:10.1002 / mrd.22387; Gandhi et al., Human Reproduction, 2000.15(2):395-401; Rizos et al., Reproductive Biology, 2003. 68(1):236-243.In some embodiments, the cell culture medium for embryo culture (including, but not limited to, any of the previously described media) is supplemented with N2, B27, FGF, and IWR-1. In some embodiments, the media are as described in Bogliotti et al., Proceedings of the National Academy of Sciences 2018.115(9), doi.org / 10.1073 / pnas.1716161115.

[0136] In some example embodiments, the number of donor exogenous cells introduced into the host is about 1 to about 25 cells, or about 30-50% of the total number of cells present in the embryo prior to the introduction of the exogenous cells. In some example embodiments, the number of exogenous cells introduced 3 or 4 days after fertilization is about 5 cells. In some example embodiments, the number of exogenous cells introduced 5 days after fertilization is 8 cells. In some example embodiments, the number of exogenous cells introduced 5 days after fertilization is 9 cells. In some example embodiments, the number of exogenous cells introduced 5 days after fertilization is 10 cells. In some example embodiments, the number of exogenous cells introduced 6 days after fertilization is about 10-20 cells.

[0137] In some example embodiments, the self-renewing exogenous donor cell is an embryonic stem cell, an extended embryonic stem cell, a pluripotent stem cell, an induced pluripotent stem cell, a multipotent stem cell, a totipotent stem cell, a primordial germ cell, a primordial germ cell-like cell, a totipotent cell, or a combination thereof.

[0138] In some example embodiments, the host non-human animal embryo into which the exogenous cells are introduced is genetically germline ablation. In some example embodiments, the non-human animal embryo comprises or is composed of one or more engineered host cells, as described elsewhere herein, of any engineered host cells. In some example embodiments, the self-renewing exogenous cell is germline capable. In some example embodiments, the self-renewing exogenous cell is an engineered cell containing one or more gene modifications in one or more target genes, and wherein said one or more target genes are not NANOS3. In some example embodiments, the self-renewing exogenous cell is not genetically modified. In some example embodiments, the self-renewing exogenous cell comprises an elite genome, a genome-selected genome, or both. Complementary embryos and animals

[0139] Animals containing both host (first cell or cell population) and donor (second cell or cell population) cells produced through the previously described complementation techniques are referred to herein as complementary animals. Such terms encompass specific cell type complementation (e.g., germ cells or other tissue type cells) and genetic complementation (e.g., specific genome or genotype complementation). It should be understood that in some cases, complementary embryos, animals, and / or their offspring may be considered non-natural or engineered, and in other cases, complementary embryos, animals, and / or their offspring may be considered natural or non-engineered. Such circumstances may be influenced by the complementary cell type and / or genetics. Complementary animals can be used as substitute male or female animals capable of producing offspring. Offspring can be obtained through any suitable method or technique, including natural mating, in vitro fertilization, artificial insemination, embryo transfer, etc.

[0140] In some exemplary embodiments herein, complementary nonhuman animal or embryos are described, comprising or consisting of a first cell population (host cells) comprising one or more cells and a second cell population (donor cells) comprising one or more cells, wherein the first cell population consists of engineered nonhuman animal cells or populations thereof as described elsewhere herein, wherein the second cell population is not any of the preceding paragraphs and / or engineered defective nonhuman cells or populations thereof as described elsewhere herein. In some exemplary embodiments, the second cell population comprises or consists of one or more engineered cell populations, wherein the one or more engineered cell populations contain one or more genetic modifications in one or more target genes, and wherein the one or more target genes are not NANOS3. In some exemplary embodiments, the second cell population is not an engineered cell or population thereof. In some exemplary embodiments, the second cell population comprises an elite genome, a genome-selected genome, or both. In some exemplary embodiments, the second cell population comprises or consists of: one or more embryonic cells, optionally fertilized eggs or inner cell mass cells; stem cells, optionally embryonic stem cells or induced pluripotent stem cells; spermatogonial stem cells or oogonial stem cells; primordial germ cells; or primordial germ cell-like cells. In some example embodiments, the second cell population is self-renewing cells. In some example embodiments, the second cell population is pluripotent, totipotent, or specialized. In some example embodiments, the second cell population is germline capable.

[0141] In some example embodiments, the complementary embryo is a morula. In some example embodiments, the complementary non-human animal or embryo is male. In some example embodiments, the complementary non-human animal or embryo is female. In some example embodiments, the complementary non-human animal or embryo is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, mouse, hare, or guinea pig.

[0142] The non-human animals described in some example embodiments herein develop from or are produced from complementary non-human animals or embryos described elsewhere herein. In some example embodiments, one or more germ cells of the non-human animal are derived from the second cell population (donor cells). In some example embodiments, approximately 0.001% to 100% of the germ cells are derived from the second cell population. In some example embodiments, the non-human animal is male. In some example embodiments, the non-human animal is female.

[0143] In some example embodiments, the complementary embryo is a preimplantation embryo, optionally a fertilized egg, a 2-cell, 4-cell, 8-cell, 16-cell, blastocyst, or morula. In some example embodiments, the first cell population comprises a percentage of the cells of the complementary nonhuman animal or embryo ranging from about 25% to at most but not including 100%. In some example embodiments, the complementary nonhuman animal or embryo contains at least one cell from the second cell population, optionally wherein the second cell population comprises a percentage of the cells of the nonhuman animal or embryo ranging from any non-zero percentage to about 75%. In some example embodiments, the complementary embryo is a day 3 post-fertilization embryo, a day 4 post-fertilization embryo, a day 5 post-fertilization embryo, or a day 6 post-fertilization embryo. In some example embodiments, the complementary embryo on day 3 post-fertilization contains about 5 cells from the second cell population; the complementary embryo on day 4 post-fertilization contains about 5 cells from the second cell population; the complementary embryo on day 5 post-fertilization contains about 8-10 cells (e.g., 8, 9, or 10 cells) from the second cell population; and the complementary embryo on day 6 post-fertilization contains about 10-20 cells from the second cell population.

[0144] In some embodiments, germline complementation produces complementary embryos and / or animals, said complementary embryos and / or animals being engineered NANOS3-deficient nonhuman animals or embryos. In addition to NANOS3-deficient cells, said complementary embryos and / or animals further comprise a second cell population containing one or more cells, said second cell population not containing any of the preceding paragraphs or engineered nonhuman animal cells as described elsewhere herein, and said second cell population being germline-capable cells, germ cells, or gametes. In some example embodiments, the second cell population comprises or consists of: one or more embryonic cells, optionally fertilized eggs or inner cell mass cells; stem cells, optionally embryonic stem cells or induced pluripotent stem cells; spermatogonial stem cells or oogonial stem cells; primordial germ cells; or primordial germ cell-like cells. In some example embodiments, the second cell population comprises or consists of one or more sperm cells or one or more oocytes. In some example embodiments, the second cell population comprises or consists of sperm or eggs. In some example embodiments, the second cell population comprises or is composed of one or more engineered cells, said engineered cells containing one or more genetic modifications in one or more target genes, wherein said one or more target genes are not NANOS3. In some example embodiments, the second cell population does not comprise or is not composed of engineered cells or a population thereof. In some example embodiments, the second cell population comprises an elite genome, a genome-selected genome, or both or composed of thereof.

[0145] This article also provides complementary embryos and / or offspring of animals. Host and donor cells for germline complementation

[0146] like Figure 1 As shown, germline complementation utilizes germline-deficient or germline-ablated host animals / cells and germline or genetic donor cells / animals. Germline-ablated host animals / cells may be NANOS3-deficient and are described in further detail below. Donor cells contain the desired genetics to be ultimately delivered in the complementation strategy by replacing male or female animals. In some embodiments, the donor animal / cell is genetically modified to contain the desired genotype and / or transgenes. In some embodiments, the donor animal / cell is not genetically modified. In some embodiments, the donor cell contains elite genetics. The donor animals and cells are described in more detail below. Engineered NANOS3-deficient host cells and organisms

[0147] Germ complementation allows for shorter generation intervals to increase the rate of genetic improvement, utilizing both unmodified and modified genetic seed stocks, even at the level of commercial animals. See, for example, Figure 1For example Figure 1 A key component of the germline complementation strategy shown and described herein is a germline-depleted host organism or embryo. Various embodiments herein describe non-human animals lacking or deficient in the functional NANOS3 gene and / or gene product, specifically cattle, their cells, and their offspring, such that the non-human animals are genetically germline-deficient / abolished. Generally, such non-human animals contain one or more genetic modifications that result in the NANOS3 gene and / or gene product being defective or eliminated, ultimately leading to a lack of sufficient function of the NANOS3 gene and / or gene product, thereby making the non-human germline-abolished, depleted, defective, and / or incompetent. Non-human animals and cells genetically modified to eliminate, deplete, lack, and / or render the NANOS3 gene and / or gene product non-functional are generally referred to herein as NANOS3-deficient cells and organisms (e.g., non-human animals). Thus, in some embodiments, NANOS3-deficient organisms (such as non-human animals) are genetically germline-deficient / abolished. Without being bound by theory, genetic germline excision / defect is caused by modification of the NANOS3 gene and / or its product, such modification rendering the NANOS3 gene and / or its product nonfunctional. NANOS3-deficient cells and / or organisms can be used to generate germline-compatible NANOS3-deficient cells, embryos, and / or adult animals through appropriate complementation strategies (see, for example, Figure 1 Further embodiments, features, and advantages of such modifications, cells, and organisms will now be described in more detail.

[0148] Engineered NANOS3-deficient cells and organisms are described in several exemplary embodiments herein. In some embodiments, the cells are bovine cells. In some embodiments, the NANOS3-deficient organisms are cattle. In some embodiments, the NANOS3-deficient organisms are male. In some embodiments, the NANOS3-deficient organisms are female. In some embodiments, the NANOS3-deficient organisms are male cattle. In some embodiments, the NANOS3-deficient organisms are female cattle. Such cells can be used in embryonic complementation strategies to supplement NANOS3-deficient embryos with allogeneic donor cells, specifically germline allogeneic donor cells. In other embodiments, NANOS3-deficient organisms can be supplemented with allogeneic cells that are depleted or absent in the NANOS3-deficient organism, such as germline cells or germline progenitor cells, or germline-capable embryonic cells. In some embodiments, NANOS3-deficient organisms can be supplemented with allogeneic cells capable of producing cells absent or depleted in NANOS3-deficient organisms, such as progenitor cells or stem cells capable of producing cells absent or depleted in NANOS3-deficient organisms. In some embodiments, the depleted or absent cells in NANOS3-deficient organisms are germline and / or germline progenitor cells. Figure 1 In this context, the NANOS3-deficient organism is the “host” animal, and the allogeneic cells that can be introduced into the NANOS3-deficient organism are “donor cells” that can be obtained from a “donor cell source” and optionally modified as described elsewhere herein.

[0149] In some example embodiments, the engineered non-human animal cells or populations thereof (e.g., host NANOS3-deficient cells or populations thereof) are bovine, horse, pig, sheep, goat, camel, deer, dog, cat, mouse, rabbit, or guinea pig cells. In some example embodiments, the engineered non-human animal cells or populations thereof are engineered somatic cells or populations thereof. In some example embodiments, the engineered non-human animal cells or populations thereof are engineered germ cells or populations thereof. In some example embodiments, the engineered germ cells or populations thereof are engineered gametes or populations thereof. In some example embodiments, the engineered gametes or populations thereof are engineered sperm or populations thereof or engineered oocytes or populations thereof. In some example embodiments, the engineered germ cells or populations thereof are engineered immature germ cells or populations thereof. In some example embodiments, the engineered immature germ cells or populations thereof are engineered sperm cells or populations thereof or engineered oocytes or populations thereof. In some example embodiments, the engineered non-human animal cells are populations of their engineered embryonic cells, optionally wherein the engineered embryonic cells are fertilized eggs. In some example embodiments, the engineered nonhuman animal cell population is an engineered blastocyst cell or a population thereof, optionally an engineered inner cell mass cell or a population thereof. In some example embodiments, the engineered nonhuman animal cell or population thereof is an engineered stem cell or a population thereof, optionally an engineered embryonic stem cell or a population thereof, or an induced pluripotent stem cell or a population thereof. In some example embodiments, the engineered nonhuman animal cell or cell population is an engineered spermatogonial stem cell or a population thereof, or an engineered oogonial stem cell or a population thereof. In some example embodiments, the engineered nonhuman animal cell or cell population is a primordial germ cell or a population thereof, or an engineered primordial germ cell-like cell or a population thereof. In some example embodiments, the engineered nonhuman animal cell or population thereof is an engineered self-renewing cell or a population thereof. In some example embodiments, the engineered nonhuman animal cell is pluripotent, totipotent, or multipotent. In some embodiments, the engineered nonhuman animal cell is a pluripotent cell as described in International Patent Application Publication WO2019 / 140260.

[0150] In some example embodiments, one or both NANOS3 alleles are modified. In some example embodiments, the engineered non-human animal cells or population thereof are monoallelic for the NANOS3 gene modification. In some example embodiments, the engineered non-human animal cells or population thereof are bialelic for the NANOS3 gene modification. In some example embodiments, the engineered non-human animal cell population does not express a functional NANOS3 gene or gene product.

[0151] In some example embodiments, the engineered non-human animal cells are heterozygous or homozygous for the NANOS3 gene modification, wherein the NANOS gene modification is optionally NANOS3 gene knockout.

[0152] In some example embodiments, the engineered nonhuman animal cells or populations thereof (e.g., NANOS3-deficient nonhuman animal cells or populations thereof) are engineered male cells or populations thereof. In some example embodiments, the engineered nonhuman animal cells are engineered female cells or populations thereof.

[0153] This document also provides engineered non-human animal embryos, engineered non-human animals, and their offspring that are NANOS3-deficient and / or have one or more engineered non-human animal NANOS3-deficient cells. The engineered NANOS3-deficient non-human animals may include one or more engineered NANOS3-deficient non-human animal cells as described herein. The offspring of NANOS3-deficient non-human animals are also described herein. Offspring can be obtained by any suitable method or technique, including natural mating, in vitro fertilization, artificial insemination, embryo transfer, etc.

[0154] In some example embodiments, the engineered nonhuman animal, embryo, or its offspring is a chimera. In some example embodiments, the engineered nonhuman animal, embryo, or its offspring is a mosaic. In some example embodiments, the engineered nonhuman animal, embryo, or its offspring is not a chimera. In some example embodiments, the engineered nonhuman animal, embryo, or its offspring is not a mosaic. In some example embodiments, at least 0.0001% to 100% of at least one cell or all cells of the engineered nonhuman animal, embryo, or its offspring are engineered nonhuman animal cells as described elsewhere herein (e.g., engineered NANOS3-deficient nonhuman animal cells).

[0155] In some embodiments, 0.0001%-100%, 0.0001%-0.001%, 0.001%-0.01%, 0.01%-0.1%, 0.1%-1%, 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% of all cells from the engineered non-human animal, embryo, or its offspring are engineered non-human animal cells (e.g., engineered NANOS3-deficient non-human animal cells).

[0156] In some example embodiments, the engineered NANOS3-deficient nonhuman animal, embryo, or its offspring is male. In some example embodiments, the engineered NANOS3-deficient nonhuman animal, embryo, or its offspring is female. In some example embodiments, the engineered NANOS3-deficient nonhuman animal is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, rat, rabbit, or guinea pig.

[0157] The engineered NANOS3-deficient nonhuman animals, embryos, or their offspring can be used as host animals in germline complementarity strategies, as described in more detail elsewhere in this document.

[0158] In some embodiments, a method for generating a NANOS3-modified nonhuman animal or embryo includes introducing one or more NANOS3 gene modifications into a nonhuman animal cell, wherein the NANOS3 gene modification reduces or eliminates the expression of the NANOS3 gene product; and one or more of the following techniques: somatic cell nuclear transfer, oocyte pronuclear DNA microinjection, zygote microinjection or embryo microinjection, intracytoplasmic sperm injection, in vitro fertilization, embryo transfer, in vitro embryo culture, or any combination thereof. NANOS3 gene modification is described elsewhere herein. In some example embodiments, introducing one or more NANOS3 gene modifications into the nonhuman animal cell comprises CRISPR-Cas-mediated gene modification, zinc finger nuclease gene modification, TALEN-mediated gene modification, recombinase-mediated gene modification, leader editing-mediated gene modification, broad-spectrum nuclease-mediated gene modification, transposase / transposon-mediated gene modification, or any combination thereof. In some example embodiments, introducing one or more NANOS3 gene modifications into the non-human animal cells involves using a CRISPR-Cas system, wherein the guide RNA of the CRISPR-Cas system targets exon 1 of the NANOS3 gene, optionally in the zinc finger region, and optionally selected from any one or any combination of SEQ ID NO:39-45. Other methods and techniques for introducing gene modifications such as NANOS3 are described in more detail elsewhere herein. NANOS3 modification

[0159] In some embodiments, one or more copies or alleles of NANOS3 may be modified to reduce (e.g., reduce to below detectable or functional levels) and / or eliminate the expression of the NANOS3 gene and / or its product. Any suitable gene or genetic modification system may be used to modify the NANOS3 gene. Exemplary genetic modification systems are described in more detail elsewhere herein.

[0160] NANOS3-deficient cells and organisms can be generated using any suitable genetic modification method and / or system. Exemplary suitable systems and working examples elsewhere in this document are described in more detail below.

[0161] By utilizing genetic modification systems, it is possible to modify polynucleotides (e.g., gene and / or transcribed gene products) encoding NANOS3 to produce germline ablation hosts, and this can occur at any appropriate stage of the system utilized. For example, modification can occur in vitro or in vitro using any suitable system (CRISPR-Cas, transposons, ZFN, TALEN, etc.) to modify polynucleotides (e.g., genomes) in fertilized eggs, developing embryos, early embryos, blastocysts, blastomeres, morulae, embryonic stem cells, primordial germ cells, primordial germ-like cells, pluripotent stem cells (including, but not limited to, cells described in some embodiments, which are pluripotent embryonic stem cells as described in International Patent Application Publication WO 2019 / 140260), induced pluripotent stem cells (e.g., cells reprogrammed from somatic cells), spermatogonial or oogonial stem cells, etc. Exemplary suitable systems for the genetic modification of polynucleotides, and more specifically, genomes, are described in more detail elsewhere herein.

[0162] The modifications in the polynucleotide encoding NANOS3 can be insertions, deletions, insertions and deletions (indels) substitutions, or any combination thereof.

[0163] In some example embodiments, the NANOS3 gene modification is the insertion of one or more nucleotides; the deletion of one or more nucleotides; the substitution of one or more nucleotides; or any combination thereof. In some example embodiments, the NANOS3 gene modification is located in exon 1, exon 2, or both of the NANOS3 gene. In some example embodiments, the NANOS3 gene modification is located in exon 1 of the NANOS3 gene, optionally within the zinc finger domain of the NANOS3 gene.

[0164] In some embodiments, the modified NANOS3 polynucleotide (gene) has a sequence that is 80%-100% identical to, at least 85% identical to, at least 90% identical to, at least 95% identical to, at least 96% identical to, at least 96% identical to, at least 97% identical to, at least 98% identical to, or at least 100% identical to any one of SEQ ID NO: 8, 10, or 11, or contains a region comprising at least 20 consecutive nucleotides. SEQ ID NO:8 >Bovine NANOS3 gene >ENSBTAG00000000399 SEQ ID NO:10 >Bos taurus NANOS3 exon 1 >ENSBTAE00000003992 ATGGGGACCTTCAACCTGTGGACAGACTACTTGGGTTTGGCACGCCTGGTTGGGGCTCAGCGTGAAGAAGAGGAGCCGGAGACCAGGCTGGATCGCCAGCCAGAAGCAGTGCCCGAACCG GGGGGTCAGCGACCCAGCCCTGAATCCTCACCAGCTCCCGAGCGCCTGTGTTCTTTCTGCAAACACAACGGCGAGTCCCGGGCCATCTACCAGTCCCACGTGCTCAAGGATGAAGCGGGCCGGGTGCTGTGCCCCATCCTCCGCGACTACGTGTGCCCCCAGTGCGGGGCCACCCGCGAGCGCGCCCACACCCGCCGCTTCTGCCCGCTCACCGGCCAGGGCTACACCTCCGTCTACAGCTACACCACCCGGAACTCGGCCGGCAAGAAGCTGGTCCGCTCGGACAAGGCGAGGACGCAGGACCCTGGACACGGACCGCGCCGAGGAGGAG SEQ ID NO:11 >Bos taurus NANOS3 exon 2 >ENSBTAE00000411199 GTTCCAAAGGTGCCAGGAAGTCTTCTGGAACTCCTCCCTCTTCCTGCTGCCCCTCAACTTCTGCCTAAGGAGACTGGCGTGGGCAGGATGACGCCTCACCTGGGGATGGGGACCCAGGCTCAGTGGAGGCTGGGTTTCAGGGAAGACCCACCCTCCGAGGATCCGCCC CCTAGACGGTGCCTCCAGCCTGGGGGCTTGGCAAAGGAGCCCGGTCTGGGACCACCGCCCAAAGCGCGCCCGCCCCTGTCACTGAAGGGGGTGGTCCTCAGGCACCCTGCCCTTCTTCCCCAACGCTGAGCAACCAGTCAGCGCTCAATAAATGTTTATGAATGGATCA

[0165] In some embodiments, the NANOS3 gene transcript (i.e., mRNA) expressed by the modified NANOS3 polynucleotide (gene) has a sequence that is 80%-100% identical to, at least 85% identical to, at least 90% identical to, at least 95% identical to, at least 96% identical to, at least 96% identical to, at least 97% identical to, at least 98% identical to, or at least 99% identical to any one of SEQ ID NO: 2, 4, 5, 7 or 9 or a region comprising at least 20 consecutive nucleotides thereof. SEQ ID NO:2 NCBI Reference Sequence: XM_027547963.1 >XM_027547963.1 predicts: nanos C2HC type zinc finger 3 (NANOS3) in zebu and common cattle, transcript variant X1, mRNA polynucleotide sequence SEQ ID NO:4 NCBI Reference Sequence: XM_027547964.1 >XM_027547964.1 predicted: zebu and common cattle (Bos indicus x Bos taurus) nanosC2HC type zinc finger 3 (NANOS3), transcript variant X2, mRNA polynucleotide sequence GGATCGCCCCCTAGACGGTGCCTCCAGCCTGGGGGCTTGGCAAAGGAGCCCGGTCTGGGACCACCGCCCAAAGCGCGCCCGCCCCTGTCACTGAAGGGGGTGGTCCTCAGGCACCCCTGCCCTTCTTCCCCAACGCTGAGCAACCAGTCAGCGCTCAATAAATGTTTATGAATGGATCAGCGTCA SEQ ID NO:5 NCBI Reference Sequence: XR_003511972.1 >XR_003511972.1 predicts: zebu and common cattle nanos C2HC type zinc finger 3 (NANOS3), transcript variant X3, RNA polynucleotide sequence 1 tgggaggcgg cggccgcggg ttcgagccgg cgccggagcc ccgcggtccc ctccccctgc 61 ccgcggcctg gggagccccc gcccagcccc ggagccgcca aaatgcaatt tcccgtgccg 121 gcgcctcgcg gctcgggggg cttttccggg cgggttttgg acagaagagg gggaaacaag 181 gcggcggccc caaaacgagg ttccaaaggt gccaggaagt cttctggaac tcctccctct 241 tcctgctgcc cctcaacttc tgcctaagga gactggcgtg ggc SEQ ID NO:7 NCBI Reference Sequence: XM_019964015.1 XM_019964015.1 predicted: zebu nanos C2HC type zinc finger 3 (NANOS3), mRNA polynucleotide sequence. 1 tccccccctc ccgccccccca aaactgctca gccagaacct gaccctgacc ctggcctttc 61 acccctcgag gagggctggt gtctggggta cttaaagaca caggctagat ttgggggcat 121 caatcctgga gggctgtgga caggaattac aagtttagga ctgggcagct gaaaaacctt 181 tctgaaaggg attagggggc cctgcttcca gaaggctcag tgaagctttc ttgaatgaat 241 gaatgaatga ggtgtgtagg cggcacgtca cctcttctct gagttccagt cttgggccct 301 gctttctcac ccttcttacc tggtacctgc agacccctcc tttaccttca gttgcccacc 361 tagcacctga tgcccgttga tcacctgcca gtctgtgtcc cacctgggtg actcgggggc 421 acaccgcatc ctcctgagat ggagcgcagg tctcatttga gagggcaatc aaggwcctgg 481 ccaatctagg ggtctcccct ctgccccgtt agccccacct gtgcctgtgc tctcttcccc 541 ataatcctca gtctcaaacc cttttccacc ccaggacctg gagagactga ctccacaaca 601 cctaaggctc ctgtaactgg tgggggaggc aggctttgtt gccttcgtga ataaccccag 661 ggcaggtgac ttcaaacccg tttgttcatc agctaaaagg aggttccact gacaaggggt 721 gtgaaagctc cctgagggtg accagaggta ggggccttgg tccttgtccc cccccaccat 781 aagacaggcc cttccctcctt ccaagtcag ctggaagttc agtggctccc cctccccct 841 cccccagtcc tggaagga agaagaagt tactaagtta ctgactacag cactgctagt 901 ctttggtg gggctccaa tgccccacc tgcatcactc tggtctct ggaggagtag 961 acaagggcag cccctcagt gcctctggg tggttgtgt ggcttatt tgctgtacc 1021 ccctgcagcc tgtgtcttgt cacgccccct cccttagc ctacccagaggccatgcagc 1081 cccgtggcag gtgcatttt gggggagct gcagcaagcc ccctgtggcaatagggaacc 1141 tcctacagcc tgctcccc tctcacacc cccttggagt ataggagggactgacagc 1201 ccagactcct cggctccaga gagggagg gagggagat taggcagaggtagagagacc 1261 agcttgggg cggctgcgtt tctcgtct tctgccctc cacctggcacacggggcca 1321 gccatgggga cttcaacct gtggacagac tactggtt tggcacgctggttggggct 1381 cagcgtgaag agaggagcc ggagaccagg ctggatcgcc agccagaagcagtgcccgaa 1441 ccggggctc agcgacccag ccctgaatcc tcaccagctc ccgagcgcctgtgttctttc 1501 tgcaaacaca acggcgagtc ccgggccatc taccagtccc acgtgctcaaggatgaagcg 1561 ggccgggtgc tgtgccccat cctccgcgac tacgtgtgcc cccagtgcggggccacccgc 1621 gagcgcgccc acacccgccg cttctgcccg ctcaccggcc agggctacacctccgtctac 1681 agctacacca cccggaactc ggccggcaag aagctggtcc gctcggacaaggcgaggacg 1741 caggaccctg gacacggacc gcgccgagga ggaggtgcct gtgcaggttccaaaggtgcc 1801 aggaagtctt ctggaactcc tccctcttcc tgctgcccct caacttctgcctaaggagac 1861 tggcgtgggc aggatgacgc cttcacctgg ggatggggac ccaggctcagtggaggctgg 1921 gtttcaggga agacccaccc tccgaggatc cgccccctag acggtgcctccagcctgggg 1981 gcttggcaaa ggagcccggt ctgggaccac cgcccaaagc gcgcccgcccctgtcactga 2041 agggggtggt cctcaggcac ccctgccctt cttccccaac gctgagcaaccagtcagcgc 2101 tcaataaatg tttatg SEQ ID NO:9 >Bos taurus Nanos3 transcript (complete) >ENSBTAT00000000513 ATGGGGACCTTCAACCTGTGGACAGACTACTTGGGTTTGGCACGCCTGGTTGGGGCTCAGCGTGAAGAAGAGGAGCCGGAGACCAGGCTGGATCGCCAGCCAGAAGCAGTGCCCGAACCGGGGGGTCAGCGACCCAGCCCTGAATCCTCACCAGCTCCCGAGCGCCTGTGTTCTTTCTGCAAACACAACGGCGAGTCCCGGGCCATCTACCAGTCCCACGTGCTCAAGGATGAAGCGGGCCGGGTGCTGTGCCCCATCCTCCGCGACTACGTGTGCCCCCAGTGCGGGGCCACCCGCGAGCGCGCCCACACCCGCCGCTTCTGCCCGCTCACCGGCCAGGGCTACACCTCCGTCTACAGCTACACCACCCGGAACTCGGCCGGCAAGAAGCTGGTCCGCTCGGACAAGGCGAGGACGCAGGACCCTGGACACGGACCGCGCCGAGGAGGAGGTTCCAAAGGTGCCAGGAAGTCTTCTGGAACTCCTCCCTCTTCCTGCTGCCCCTCAACTTCTGCCTAAGGAGACTGGCGTGGGCAGGATGACGCCTTCACCTGGGGATGGGGACCCAGGCTCAGTGGAGGCTGGGTTTCAGGGAAGACCCACCCTCCGAGGATCCGCCCCCTAGACGGTGCCTCCAGCCTGGGGGCTTGGCAAAGGAGCCCGGTCTGGGACCACCGCCCAAAGCGCGCCCGCCCCTGTCACTGAAGGGGGTGGTCCTCAGGCACCCCTGCCCTTCTTCCCCAACGCTGAGCAACCAGTCAGCGCTCAATAAATGTTTATGAATGGATCA

[0166] In some embodiments, the modified NANOS3 polynucleotide (gene) encodes a NANOS3 polypeptide that is 80%-100% identical to any one of SEQ ID NO: 1, 3, or 6; at least 85%; at least 90%; at least 95%; at least 96%; at least 96%; at least 97%; at least 98%; at least 99%; 85%-100%; 90%-100%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; 99%; or 100% identical. SEQ ID NO: 1 NCBI Reference Sequence: XM_027547963.1 >XM_027547963.1 predicts: zebu and common cattle nanos C2HC type zinc finger 3 (NANOS3), transcript variant X1, mRNA polypeptide sequence MGTFNLWTDYLGLARLVGAQREEEEPETRLDRQPEAVPEPGGQRPSPESSPAPERLCSFCKHNGESRAIYQSHVLKDEAGRVLCPILRDYVCPQCGATRERAHTRRFCPLTGQGYTSVYSYTTRNSAGKKLVRSDKARTQDPGHGPRRGGGACAGSKGARKSSGTPPSSCCPSTSA SEQ ID NO:3 NCBI Reference Sequence: XM_027547963.1 MGTFNLWTDYLGLARLVGAQREEEEPETRLDRQPEAVPEPGGQRPSPESSPAPERLCSFCKHNGESRAIYQSHVLKDEAGRVLCPILRDYVCPQCGATRERAHTRRFCPLTGQGYTSVYSYTTRNSAGKKLVRSDKARTQDPGHGPRRGGGSKGARKSSGPPSSCCPSTSA SEQ ID NO:6 NCBI Reference Sequence: XM_019964015.1 MGTFNLWTDYLGLARLVGAQREEEEPETRLDRQPEAVPEPGGQRPSPESSPAPERLCSFCKHNGESRAIYQSHVLKDEAGRVLCPILRDYVCPQCGATRERAHTRRFCPLTGQGYTSVYSYTTRNSAGKKLVRSDKARTQDPGHGPRRGGGACAGSKGARKSSGTPPSSCCPSTSA

[0167] In some embodiments, the modified NANOS3 gene corresponds to 80% to 100% of the bovine NANOS3 gene sequence (accession number NC007305.5, region 10061880) in the National Center for Biotechnology Information database (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4 ... (6.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% to / or 100%) are the same.

[0168] In some embodiments, the modified NANOS3 gene is a homolog, ortholog, or paralog of the bovine NANOS3 gene and / or the NANOS3 sequence of this disclosure. In some embodiments, the NANOS3 homolog consists of or includes a nucleotide sequence, said nucleotide sequence being, for example, but not limited to, at least 80%; at least 85%; at least about 90%; at least about 91%; at least about 92%; at least about 93%; at least about 94%; at least about 95%; at least about 96%; at least about 97%; at least about 98%; at least about 99%; at least about 99.5%; 99.6%, 99.7%, 99.8%, and / or at least about 99.9% identical to about 20 consecutive nucleotides in one or more NANOS3 sequences of this disclosure.

[0169] Site-specific modification of the endogenous NANOS3 gene in host cells and / or animals results in the disruption of the NANOS3 gene and / or gene / product and / or its expression, which can be achieved by any suitable technique, such as any technique described elsewhere herein. Generally, such methods involve contacting cells with one or more genetic modification systems described herein, said genetic modification systems being configured to modify the NANOS3 gene, specifically the bovine NANOS3 gene or components thereof. In some embodiments, the system employs site-specific modification of the NANOS3 gene using natural homolog recombination pathways (such as conventional knock-in and knock-out methods that rely on homologous arms to guide site-specific knock-in of disruptive exogenous polynucleotides) and others such as RNA-guided nucleases (e.g., CRISPR-Cas) and transposons. Examples of suitable genetic modification systems and techniques are described in more detail herein, and those skilled in the art will understand such suitable genetic modification systems and techniques in light of this disclosure.

[0170] In some embodiments, a CRISPR-Cas-based approach is used to modify the NANOS3 gene to introduce substitutions, indels, or other mutations, thereby effectively reducing or eliminating NANOS3 gene function and the production of NANOS3 gene products. Exemplary wizards for CRISPR-Cas9 NANOS3 knockout are provided at least in the working examples below and can be designed based on the principles and descriptions provided in this disclosure. In some embodiments, the number of modified, substituted, inserted, and / or deleted nucleotides may be, or in total (in the case of indels), 1-2600 or more. In some embodiments, NANOS3 is modified at one or more nucleotides in exon 1, exon 2, or both.

[0171] In some embodiments, the number of modified, substituted, inserted, and / or deleted nucleotides may be or total (in the case of indel) approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 34 of the NANOS3 gene. 0, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 5 90, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 8 40, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 107 0, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 12 80, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1 490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1630, 1640, 1650, 1660, 1670, 1680, 16901700, 1710, 1720, 1730, 1740, 1750, 1760, 1770, 1780, 1790, 1800, 1810, 1820, 1830, 1840, 1850, 1860, 1870, 1880, 1890, 1900, 1910, 1920 1930, 1940, 1950, 1960, 1970, 1980, 1990, 2000, 2010, 2020, 2030, 2040, 2050, 2060, 2070, 2080, 2090, 2100, 2110, 2120, 2130, 2140, 2150 2160, 2170, 2180, 2190, 2200, 2210, 2220, 2230, 2240, 2250, 2260, 2270, 2280, 2290, 2300, 2310, 2320, 2330, 2340, 2350, 2360, 2370, 2380 2390, 2400, 2410, 2420, 2430, 2440, 2450, 2460, 2470, 2480, 2490, 2500, 2510, 2520, 2530, 2540, 2550, 2560, 2570, 2580, 2590, 2600 or more nucleotides.

[0172] In some embodiments, NANOS3 polynucleotide modification results in a decrease in the expression of the NANOS3 gene and / or its product by approximately 1 to 1000-fold or more. In some embodiments, NANOS3 modification results in a decrease in the expression of the NANOS3 gene and / or its product by approximately 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 2... 10 times, 220 times, 230 times, 240 times, 250 times, 260 times, 270 times, 280 times, 290 times, 300 times, 310 times, 320 times, 330 times, 340 times, 350 times, 360 times, 370 times, 380 times, 390 times, 400 times, 410 times, 420 times, 430 times, 440 times, 450 times, 460 times, 470 times, 4 80 times, 490 times, 500 times, 510 times, 520 times, 530 times, 540 times, 550 times, 560 times, 570 times, 580 times, 590 times, 600 times, 610 times, 620 times, 630 times, 640 times, 650 times, 660 times, 670 times, 680 times, 690 times, 700 times, 710 times, 720 times, 730 times, 740 times 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, or more times. In some embodiments, NANOS3 polynucleotide modification results in the expression level of the NANOS3 gene or gene product being unobservable or undetectable. In some embodiments, NANOS3 polynucleotide modification results in a reduction or complete elimination of germ cells in animals having modified NANOS3, wherein the reduction results in the substantial absence of germ cells in animals having modified NANOS3. Methods for measuring gene and gene product expression include, but are not limited to, PCR-based techniques and affinity and immunological protein detection methods, which are generally known in the art.

[0173] In some embodiments, the modification reduces the expression of the NANOS3 gene or its product by 1 to 1000-fold or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 times. 170x, 180x, 190x, 200x, 210x, 220x, 230x, 240x, 250x, 260x, 270x, 280x, 290x, 300x, 310x, 320x, 330x, 340x, 350x, 360x, 370x, 380x, 390x, 400x, 410x, 420x, 430x, 440x, 45 0x, 460x, 470x, 480x, 490x, 500x, 510x, 520x, 530x, 540x, 550x, 560x, 570x, 580x, 590x, 600x, 610x, 620x, 630x, 640x, 650x, 660x, 670x, 680x, 690x, 700x, 710x, 720x, 730x 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 to / or 1000 or more times. In some embodiments, the modification reduces the expression of the NANOS3 gene or gene product such that the amount is undetectable by conventional techniques for measuring the amount of gene and / or gene product (e.g., transcript and / or protein) (e.g., below the detection limit). In some embodiments, the modification reduces the expression of the NANOS3 gene or gene product such that one or more functions or activities of the NANOS3 gene or gene product are insufficient to achieve the function or activity of the normal or wild-type NANOS3 gene or gene product, such as producing immature and mature germ cells. Therefore, in some embodiments, the NANOS3-deficient animals described elsewhere herein may be germline ablation (i.e., lacking functional germ cells) but still possess detectable expression of the NANOS3 gene or gene product. Donor cells for germline complementation Engineered donor cells

[0174] In some embodiments, the genetic modification system can be used to modify donor cells or to generate donor cell sources for germline complementation with a NANOS3-deficient host to produce alternative male and / or female animals. In some embodiments, such exogenous gene constructs are introduced into donor cells (see, for example...). Figure 1 In the donor cells, the donor cells can be used to supplement the germline ablation of NANOS3-deficient host or host cells described herein.

[0175] Exemplary donor cells and cell sources include, but are not limited to, fertilized eggs, developing embryos, early embryos, blastocysts, blastomeres, morulae, embryonic stem cells, primordial germ cells, primordial germ cell-like cells, pluripotent stem cells (including, but not limited to, the cells described in some embodiments, which are pluripotent embryonic stem cells as described in International Patent Application Publication WO 2019 / 140260), induced pluripotent stem cells (such as cells reprogrammed from somatic cells), spermatogonial or oogonial stem cells, somatic cells, tissues, organs (such as testes or ovaries), and any combination thereof.

[0176] In some embodiments, donor cells or cell sources are modified to contain and / or express exogenous engineered genes, such as any exogenous engineered genes described in U.S. Patent Application Publication No. 2019 / 165465. In some embodiments, where the exogenous cell source (e.g., an organism) contains and / or expresses engineered genes or gene constructs that result in the excision, elimination, and / or dysfunction of undesirable germline cells, it should be understood that germline donor cells from such donor cell sources used to complement this document as NANOS3-deficient hosts will not contain engineered constructs because such constructs result in the excision or rendering of germ cells expressing such constructs infertile or unable to produce viable zygotes or embryos. Such undesirable germline cells are cells carrying undesirable genes, alleles, and / or chromosomes. Exemplary undesirable genes, alleles, and / or chromosomes include disease-causing genes, alleles, and / or chromosomes (i.e., genes, alleles, and / or chromosomes that transmit genetic diseases or make animals susceptible to developing diseases or symptoms), genes, alleles, and / or chromosomes that transmit undesirable phenotypes or traits, and / or X or Y chromosomes. In some embodiments, such donor cells are not modified prior to germline complementation for use in NANOS3-deficient hosts. In some embodiments, such donor cells may be modified prior to germline complementation for use in NANOS3-deficient hosts. Exemplary modifications are described below.

[0177] In some embodiments, genetic modifications in engineered donor cells result in increased or decreased expression of one or more genes and / or gene products.

[0178] In some embodiments, donor-derived cells are modified prior to use in complementing a NANOS3-deficient host. Such modifications may include genetic modifications that can be introduced via the genetic modifications or modification systems described herein. In some embodiments, donor-derived embryos, blastomeres, or ESCs are genetically modified prior to complementation (see, for example, Figure 1 In some embodiments, donor spermatogonial (or oogonial) stem cells, pluripotent or induced pluripotent stem cells (including, but not limited to, pluripotent or induced pluripotent stem cells as described in International Patent Application Publication WO 2019 / 140260). In some embodiments, the cells are pluripotent embryonic stem cells, primordial germ cells, or primordial germ cell-like cells that have been genetically modified prior to complementation as described in International Patent Application Publication WO 2019 / 140260 (see, for example, Figure 1 ). Exemplary Gene Modifications and Transgenics of Donor Cells Modifications that produce Polled Bovines

[0179] Genes and polynucleotides can be modified to produce hornless (hornless) animals (such as hornless cattle).

[0180] In some embodiments, the modification is located proximal to bovine chromosome 1 (BAT01), optionally corresponding to the HAS21 region, and optionally begins at about bp 1,684,495 and ends at about bp 1,896,112. In some embodiments, the modification occurs in one or more genes located proximal to bovine BAT01. In some embodiments, the genome is modified to contain 90%-100% identical polynucleotides to any one of SEQ ID NO: 1-148 of U.S. Patent Publication 20110262909, and one or more polymorphisms or SNPs associated with the hornless phenotype include, but are not limited to, any one or more of the polymorphisms or SNPs shown in Tables 1, 2, or 3 of U.S. Patent Publication 20110262909. In some embodiments, the genes IFNAR2m, SYNJ1, and C21orf63 are modified to contain genotypes corresponding to the hornless phenotype (see, for example, Table 3 of U.S. Patent Publication 20110262909). In some embodiments, the PAXBP1 gene is modified to contain one or more mutations that produce the hornless phenotype. In some embodiments, the C1H21orf62 gene is modified to contain one or more mutations that produce a hornless phenotype. In some embodiments, intron 3 of the IFGR2 gene is modified to contain one or more mutations that produce a hornless phenotype, such as the SNP described in Glatzer et al., PLOS ONE 8:e67992 (2013). In some embodiments, the FOXL2 and / or RXFP2 genes are modified to contain one or more mutations that produce a hornless phenotype. In some embodiments, the ZEB2 gene is modified to contain one or more mutations that produce a hornless phenotype (see, for example, Capital et al., PLOS ONE 7:e49084). In some embodiments, the OLIG1 and / or OLIG2 genes are modified to introduce hornless mutations, thereby producing a hornless phenotype. In some embodiments, the genome is modified to introduce one or more mutations in long non-coding RNA (LNcRNA) number 1 (which maps to the annotated bovine locus LOC100848368) and LNcRNA number 2 (which overlaps with the annotated LOC100848215 or four annotated exons of a regulatory molecule), resulting in overexpression of LNcRNA number 1 and / or number 2 and the production of hornless animals. See also Allais-Bonnet et al., PLOS ONE 8:e63512 (2013). In some embodiments, one or more modifications are made to the annotated loci LOC100848368 and / or LOC10084821, such as one or more modifications that result in reduced expression of one or more gene products produced by one or both loci, resulting in a hornless phenotype.

[0181] In some embodiments, the hornless phenotype is introduced by modifying the genome to introduce three SNPs and / or an 80 kb repeat of chromosome 1 corresponding to bp1,909,352-1,989,480 (BTA1 (or BTAO1)) of chromosome 1, said chromosome being the same as the P identified in the Friesian breed in Holstein. F Allele correspondence (as described in Medugorac et al., PLOS ONE 7:e39477 (2012)). In some embodiments, a hornless phenotype is introduced by modifying the genome such that the hornless phenotype contains a 202 bp insertion-deletion consisting of a 212 bp repeat of BTA1: 1,705,834–1,706,045 bp, in place of a 10 bp deletion of BTA1: 1,706,051–1,706,060 bp or a 202 bp InDel consisting of a 208 bp repeat combined with a 6 bp deletion in BTA1, which correspond to the P identified in the Celtic original breed. C Alleles or P C Allele variants corresponding to (P) C The allele contains 202-bp InDel, resulting in a hornless phenotype. See also Wiedemar et al., PLOS ONE 9:e93435 (2014). In some embodiments, the genome is modified such that BAT1 is modified to contain the same allele as P. 219ID The 219bp repeat-insertion corresponding to the allele or its equivalent (see, for example, Medugorac et al., 2017. Nature Genetics 49:470. doi:10.1038 / ng.3775). In some embodiments, the genome is modified such that BAT1 is modified to contain a repeat corresponding to P. 1ID Alleles or their equivalents corresponding to 7bp deletions and 6bp insertions (see, for example, Medugorac et al., 2017. Nature Genetics 49:470. doi:10.1038 / ng.3775). In some embodiments, the genome is modified such that BAT1 is modified to include P G The alleles correspond to approximately 110 kb repeats. (See, for example, Stafuzza et al., (2018) PLOS ONE 13:e0202978 and Utsunomiya et al., (2019) Anim.Genet. 50, 187-188).

[0182] In some embodiments, the genome is modified such that it contains one or more sequence variants and / or SNPs from Table S1 or Table S2 of Wiedemar et al., PLOS ONE 9:e93435 (2014) that result in a hornless phenotype. Table S2 of Wiedemar et al., PLOS ONE 9:e93435 (2014) is reproduced below.

[0183] In some embodiments, modifications are introduced into the genome to produce animals with stubble horns, which produce effective hornless animals, such as cattle. In some embodiments, a genomic modification is introduced at the stubble horn (Sc) locus. In some embodiments, a genomic modification for producing cattle with a stubble horn phenotype is introduced into chromosome 19 (BTA 19). In some embodiments, a genomic modification for producing cattle with a stubble horn phenotype is introduced into the TWIST1 gene. See, for example, Berriere et al., Animal Genetics 2004.35:34-39 and Capital et al., PLOS ONE 2011.6(7):e22242.

[0184] In some embodiments, the genome is modified such that it contains one or more SNPs associated with a hornless phenotype, including but not limited to any one or more hornless phenotypes shown in U.S. Patent Application Publication 20050153328, particularly the hornless phenotypes described in paragraphs

[0036] ,

[0081] -

[0082] ,

[0088] , Tables 1 and 2, or any of the SNPs or regions that can be identified using the primers in Table 1 of U.S. Patent Application Publication 20050153328. In some embodiments, the genome is modified such that it contains one or more SNPs associated with a hornless phenotype, including but not limited to any one or more hornless phenotypes shown in U.S. Patent Application Publication 20110195414, particularly the hornless phenotypes described in paragraphs

[0013] -

[0028] ,

[0030] ,

[0033] ,

[0097] ,

[0112] , Tables 1-5.

[0185] In some embodiments, the genome is modified such that it contains SNPs or haplotypes corresponding to SNPs or haplotypes, including but not limited to any one or more of the SNPs or haplotypes described in U.S. Patent No. 8,105,776, particularly... Figure 1-42 The SNPs or haplotypes in columns 3 (3:19-67), 4:1-67, 5:1-58, 7:19-67, 8:1-67, 9:1-12, 32:40-59, 33:15-29; 33:45-50, 33:58-67, 34:1-4, 34:37-47, 35:8-13, 35:19-29, 35:35-40 and Table 1-7 are listed.

[0186] In some embodiments, the genome is modified such that it contains modifications for producing a hornless phenotype, including but not limited to any one or more of the hornless phenotypes shown in U.S. Patent Application Publication 2014 / 0201857, particularly in paragraphs

[0026] -

[0029] ,

[0035] -

[0036] ,

[0038] -

[0040] ,

[0091] -

[0098] and Figure 2 The angular-free phenotype at -5. Example modifications to improve disease resistance or tolerance

[0187] In some embodiments, the genome is modified to give the animal (e.g., cattle) improved disease resistance or tolerance. Examples of diseases in which resistance or tolerance is advantageous include, but are not limited to, mastitis, Johne's disease, bovine viral diarrhea-related diseases, and other viral and microbial mediated infections (e.g., tuberculosis, chlamydiosis, leptospirosis, campylobacterosis, salmonellosis, listerosis, myersiniosis, Pseudomonas, Aerobactor, Klebsiella, Mannhemia, Pasteurella, Histophilis, cryptosporidiosis, Escherichia coli, rabies, anthrax, antibiotic-resistant Staphylococcus (e.g., MRSA), and Fusobacterium necrophorum). (e.g., necrophorum, streptococcus, corynebacterial, various fungi), BSE, BRD, IARS syndrome, milk fever, shipping fever, grass tetany, hydrocyanic acid poisoning, white muscle disease, IBR, foot and mouth disease, foot rot, etc.)

[0188] Inactive or insufficient expression of the Peg3 gene can lead to disease intolerance and other problems. See, for example, U.S. Patent Publication 20030018987. In some embodiments, genomic modifications for improving disease resistance (such as resistance to mastitis and other diseases) include one or more modifications to the Peg3 gene to restore healthy (non-disease-related) or wild-type gene activity. In some embodiments, the modification alters a defective, inactive, or otherwise deficient Peg3 gene to an active, functional Peg3 gene. In some embodiments, the genomic modifications for improving disease resistance (such as resistance to mastitis) are located in one or more genes on chromosomes BTA5, BTA6, BTA7, BTA12, BTA13, BTA16, BTA18, BTA19, and BTA20, in any gene and / or location on such chromosomes as shown in U.S. Patent Application Publication 20150240308, particularly in

[0066] -

[0104] and

[0185] -

[211] . Figure 17The genomic modifications described in Tables 6, 2, 3, 4, 8-26 and SEQ ID NO: 1-3, and any genomic modifications described in U.S. Patent Application Publication 20110023158, including but not limited to any one or more of the genomic modifications described in paragraphs

[0047] -

[0051] .

[0189] In some embodiments, genomic modifications for improving disease resistance (such as resistance to mastitis and / or other diseases) include one or more modifications to the β-casein gene (CSN2), including but not limited to one or more modifications set forth in U.S. Patent Application Publication 20090013419, particularly the modification at paragraph

[0014] .

[0190] In some embodiments, genomic modifications for improving disease resistance (such as resistance to tuberculosis and / or other diseases) include one or more modifications of SFTPA1 and MAT1A, the intergenic region of the SP110 nuclear histone gene, the IPR1 gene, and / or the intergenic region between the FSCN1 and ACTB genes, such as the modifications shown in Chinese Patent 104293833 or Chinese Patent Application Publication CN201810813577.2A.

[0191] In some embodiments, genomic modifications for improving disease resistance include one or more modifications to any target gene that is a target gene and / or whose gene product is listed in the table of U.S. Patent No. 10,106,621, from column 6, row 25 to column 13, row 52.

[0192] In some embodiments, genomic modifications for improving disease resistance (such as resistance to bovine respiratory disease (BRD) and / or mastitis) include one or more modifications to the granulocyte colony-stimulating factor gene (G-CSF), such as any of those modifications (or analogues thereof) described in U.S. Patent No. 10,138,283, particularly in columns 27: rows 15-45 and in U.S. Patent No. 5,416,195, particularly in columns 2: rows 11-68, columns 3: rows 1-16, columns 5: rows 59-68, and columns 6: rows 1-35.

[0193] In some embodiments, genomic modifications for improving disease resistance (such as resistance to foot-and-mouth disease virus (FMDV)) include one or more modifications to the eIF4G gene, such as any modifications shown in U.S. Patent No. 10,058,078, particularly in columns 4: rows 60-67, columns 5: rows 1-50, columns 6: rows 16-53, columns 7: rows 1-40, columns 24: rows 66-67, columns 25: rows 1-30, Tables 1 and 2. Figure 2 Modifications at the location.

[0194] In some embodiments, genomic modifications for improving disease resistance (such as resistance to bovine spongiform encephalopathy (BSE or mad cow disease)) include one or more modifications, such as any of the modifications described in U.S. Patent Application Publication 20110023158, particularly one or more of the modifications described in paragraphs

[0057] -

[0059] .

[0195] In some embodiments, genomic modifications for improving disease resistance (such as resistance to mastitis and other diseases) include Peg3, SOX5, ETNK1, LOC520387, PLCZ1, PIK3C2G, RERGL, LMO3, MGST1, SLC15A5, IGJ, UTP3, RUFY3, GRSF1, MOB1B, DCK, SLC4A4, GC, NPFFR2, ADAMTS3, CAD26, EDN3, RAB22A, TMEM74B, TBC1D20, DEFB129, DEFB119, DEFB117, and DEFB. Modifications of 122a, DEFB122, DEFB123, DEFB124, ID1, XKR7, BPIFB2, BPIFB6, BPIFB3, BPIFB4, LAD1, CSRP1, MMP23B, TNFRSF4, TNFSRF18, ISG15, PLEKHN1, B3GALT6, SEC14-like protein 1, N-acetylglucosamine transferase, acetylglucosamine transferase isoenzyme B, LIFR, EP4R, complement component C9, OSMR, complement component C7 precursor, complement component C6 precursor, β-casein (CSN2), sFTPA1, MATA1A, SP110 nuclear histone gene, eIF4, or any combination thereof. Modifications for the prevention of genetic diseases

[0196] In some embodiments, the genome is modified to contain one or more modifications to prevent one or more genetic diseases. Exemplary bovine genetic defects or conditions that can be prevented through genome modification include, but are not limited to, α(α) and / or β(α)-mannosin storage disease, multiple joint contractures (AM), contracted arachnodactyly (CA), neuropathic hydrocephalus (NH), hairlessness (hairless calves), idiopathic epilepsy, osteosclerosis, protoporphyria, pulmonary hypoplasia and generalized edema (PHA), tibial hemiplegia (TH), achondroplasia (bulldog dwarfism), alopecia, joint stiffness, joint contractures (palatinopalatine syndrome, joint rigidity), brachynathia inferior (maxillary protrusion), cryptorchidism, dermoid cysts, dipymyopathy, fawn calf syndrome, hypertrichosis (rat tail), cerebral spinal cord edema (maple syrup diabetes), oculodermal hypopigmentation, polydactyly, and progressive bovine myeloencephalymprolonged gestation. gestation), syndactyly (mule foot), translocation, bovine leukocyte adhesion defect, complex spinal deformity, freemartinism, etc. See also Ciploch et al., Genes. Genomics. 2017 39(5):461-471.

[0197] In some embodiments, the genome is modified to contain one or more modifications that can prevent dwarfism or mannoside storage disease, including but not limited to any one or more of the modifications described in U.S. Patent Application Publication 20110023158, particularly one or more of the modifications described in paragraphs

[0075] -

[0078] . In some embodiments, the genome is modified to contain one or more modifications for the prevention of bovine genetic diseases, such as modifications in any gene for the prevention of any of the diseases described in Cieplch et al., Genes and Genomics 39:461-471 (2017), particularly the modifications in Table 1. Heat resistance

[0198] In some embodiments, heat or cold tolerance in animals is advantageous. In some embodiments, the genome is modified to contain one or more modifications that confer heat tolerance and / or cold tolerance (generally referred to herein as heat tolerance) to the modified animal (e.g., a cow). In some embodiments, genomic modifications for improving heat tolerance include one or more modifications to the prolactin receptor (PRLR) gene, including but not limited to any modifications shown in U.S. Patent Application Publication 201902223417, particularly in paragraph

[0008] and / or the modifications in U.S. Patent Application Publication 20170079251, particularly in paragraphs

[0006] -

[0015] ,

[0126] -

[0129] ,

[0132] ,

[0140] -

[0143] , Tables 1, 2, 4, 7-8, and Figure 1-3 Modifications at the location. Modifications to the yield or characteristics of meat or dairy products

[0199] In some embodiments, it is advantageous to modify animals (such as cattle) to give them modified and / or improved yields or characteristics of meat, milk, or other products, such as reduced allergen content, reduced lactose content, improved nutritional properties, increased marbling, or other qualities. In some embodiments, the genome is modified such that it contains one or more modifications in one or more genes, resulting in improved and / or modified yields and / or other characteristics of the meat, milk, or other products. Modifications to reduce milk allergens

[0200] Milk, particularly cow's milk, contains proteins that can be allergens in humans. The main allergenic proteins in milk, particularly cow's milk, are casein (αS1, αS2, β, κ, etc.), α-lactalbumin, and β-lactoglobulin. See, for example, Shoormasti et al., *Iran J Allergy Asthma Immunol.*, April 2016; 15(2):161-5. Other proteins in milk, such as lactoferrin, bovine IgG (e.g., IgG heavy chain), and bovine serum albumin, may also cause allergies. In some embodiments, the genome is modified to contain one or more modifications to one or more genes encoding one or more allergenic proteins in milk, particularly cow's milk, not limited to casein (αS1, αS2, β, κ, etc.), α-lactalbumin, β-lactoglobulin, lactoferrin, bovine IgG (e.g., IgG heavy chain), bovine serum albumin, or any combination thereof, such that the allergen content in the milk is reduced or eliminated.

[0201] In some embodiments, the genome is modified to contain one or more modifications that reduce one or more milk allergens, said milk allergens being the milk of a modified animal (such as a cow), wherein said modifications are located in the β-lactoglobulin gene, such as one or more genetic mutations (single or double mutations) that confer amino acid mutations in the β-lactoglobulin polypeptide (such as C160S), said one or more genetic mutations including, but not limited to, the genetic mutations shown in U.S. Patent No. 6,677,433, particularly the genetic mutation at positions 67:40-51. In some embodiments, such mutations result in milk with reduced allergen content or potential.

[0202] In some embodiments, the genome is modified to contain one or more modifications that reduce one or more milk allergens (such as proteins, lipids, fatty acids, etc.), milk from modified animals (such as cows), such as one or more genetic mutations that confer amino acid mutations in milk proteins, lipids, and / or fatty acids, including but not limited to the genetic mutations shown, for example, in U.S. Patent Application Publication 20110023158, and / or any one or more of proteins, lipids, fatty acids, etc., particularly the genetic mutations described in paragraphs

[0017] -

[0039] . Modifications to improve the nutritional properties of meat or dairy products

[0203] In some embodiments, genomic modifications result in modified nutrition or nutritional properties of meat or milk produced from animals (such as cattle) having said genetic modifications. Such modifications to nutritional properties can produce improved meat or milk that provides some nutritional or other health benefits to one or more human or animal populations that consume the meat or dairy products. Examples include, but are not limited to, milk with altered fat content, reduced lactose, or lactose-free qualities.

[0204] In some embodiments, the genome is modified to contain one or more modifications that can alter the nutritional or nutritional characteristics of the meat and / or milk of the modified animal (such as a cow), including but not limited to one or more genetic modifications described in U.S. Patent Application Publication 20110023158, particularly one or more of the modifications described in paragraphs

[0036] -

[0043] .

[0205] In some embodiments, the genome is modified to reduce the amount of lactose in the milk of a modified animal (such as a cow), including but not limited to one or more genetic modifications described in U.S. Patent Application Publication 20110023158, particularly one or more of the genetic modifications described in paragraphs

[0040] -

[0043] .

[0206] In some embodiments, the genome is modified to contain one or more modifications that can increase or alter the content of bioactive proteins in milk, including but not limited to any of the modifications described in U.S. Patent Application Publication 20110023158, particularly one or more of the modifications described in paragraphs

[0047] -

[0051] . Modifications that increase or alter the yield and / or quality of milk and meat

[0207] In some embodiments, genomic modifications result in animals (such as cattle) having increased or otherwise improved milk and / or meat production and / or carcass quality.

[0208] In some embodiments, the genome is modified to contain one or more modifications that can alter the milk and / or meat yield of the modified animal, including but not limited to one or more modifications described in U.S. Patent Application Publication 20110023158, including but not limited to one or more modifications described in paragraphs

[0044] -

[0046] ,

[0052] -

[0056] and

[0059] , one or more modifications described in U.S. Patent Application Publication 20180296522, particularly the modification in paragraph

[0015] , one or more modifications in the DGAT gene, one or more modifications described in U.S. Patent Application Publication 20060172329, particularly the modifications in paragraphs

[0007] ,

[0009] ,

[0012] ,

[0016] ,

[0077] , Tables 1-2, or any combination thereof.

[0209] In some embodiments, the genome is modified to contain one or more modifications that can alter at least one or more growth, milk and / or meat yield, milk and / or carcass traits and / or quality, including but not limited to any one or more modifications in the NCAPG gene shown in U.S. Patent Application Publication 20090260095, particularly the modification at paragraph

[0008] , any one or more modifications in the IGF-2 gene shown in U.S. Patent Application Publication 20070026404, particularly the modifications at paragraphs

[0103] -

[105] , and any one or more modifications in the FABP4 gene shown in U.S. Patent Application Publication 20070020658, particularly the modification at paragraph

[0157] . Any one or more modifications in the TFAM gene shown in Publication 20070065843, particularly the modifications in paragraphs

[0097] -

[0100] ,

[0130] ,

[0155] ,

[0164] -

[0165] ,

[0216] -

[0218] , Tables 2, 4-5; any one or more SNPs in the TFAM, TFB1M, TFB2M and / or other genes shown in U.S. Patent Application Publication 20080183394, particularly the SNPs in paragraphs

[0025] -

[00030] ,

[0096] -

[0099] ,

[0108] -

[0109] ,

[0129] ,

[0182] -

[0185] ,

[0216] -

[0219] ,

[0226] , Tables 2, 4-5. Figure 3 The SNPs at Figure 4; any one or more modifications of the modifications shown in U.S. Patent 6,383,751, particularly the modifications in column 24: row 59 to column 25: row 7 and Tables 5-8; any one or more modifications of the modifications shown in U.S. Patent Application Publication 20070026404, particularly the modifications in paragraphs

[0030] -[0044,

[0096] -

[0097] ,

[0103] ,

[0106] ; any one or more modifications of the modifications in the UCN3 gene shown in U.S. Patent 7,662,567, particularly in the abstract, column 4: 26-55, column 16: 4-46, column 18: 6-4, column 29: 47-67, column 30: 1-67, column 31: 1-22 and columns 47-67, 32: 1-22, Table 1 and Figure 1-4DModifications at the specified locations; any one or more modifications in the CRH gene as shown in U.S. Patent 7,662,567, particularly in the abstract, column 3: rows 25-28, column 4: rows 51-67, column 5: rows 1-22, column 16: rows 35-52, column 28: rows 33-62, column 31: rows 7-51, column 32: rows 1-52, column 33: rows 16-32 and 63-67, column 34: rows 64-67, column 35: rows 1-34, Table 1 and Figure 1 , 2 Modifications at A-2D, 3A-3C, and 4A-4C; any one or more of the modifications shown in U.S. Patent 8,008,011, particularly in the abstract, column 4: lines 3-31 and 48-64, column 6: lines 63-67, column 7: lines 1-22, column 8: lines 1-67, column 9: lines 1-2, Tables 2-4, and Figure 1 Modifications at -4; any one or more modifications in the leptin and / or ob genes shown in U.S. Patent Application Publication 20030219819, particularly the modifications at paragraphs

[0005] -

[0009] ,

[0037] , and

[0052] ; any one or more modifications in the UQCRC1 gene shown in U.S. Patent No. 7,879,552, particularly in columns 2: rows 40-67, columns 3: rows 10-24, columns 5: rows 18-21, columns 16: rows 6-37, columns 29: rows 13-41, columns 30: rows 15-41, Tables 1-5 and Figure 3 Modifications at A-3D; any one or more of the modifications shown in U.S. Patent No. 7,157,231, particularly the modifications in column 2: rows 6-25 and 51-67, column 3: rows 10-22 and 61-67, column 4: rows 1-19 and Tables 1-3; modifications in the adnectin gene, engineered adnectin gene or the product that produces adnectin or engineered adnectin gene; any one or more of SNPs or variants, as shown in U.S. Patent Application Publication 20190307855, particularly those in paragraphs

[0010] -

[0039] , Tables 1-4; or any combination thereof.

[0210] In some embodiments, the genome is modified to contain one or more modifications of any of the genes shown in Tables 1, 2 and / or 3 and / or those corresponding to SEQ ID NO: 1-408 in U.S. Patent No. 7,638,275.

[0211] In some embodiments, the genome is modified to contain one or more modifications in any of the genes to improve nutrition and / or treatment as shown in Wall et al., 1997. Journal of Dairy Science 80:2213-2224, particularly at Table 6. Modification of traits related to production and / or management

[0212] In some embodiments, genomic modifications endow animals (such as cattle) with improved traits associated with production and / or management, including but not limited to temperament, coat color, hair loss, hoof angle, growth, feed efficiency, lameness, blood pressure, etc. Exemplary modifications described elsewhere herein, such as heat tolerance or disease resistance, can also improve production and / or management.

[0213] In some embodiments, the genome is modified to contain one or more modifications that can alter the coat color or other coat color (such as hair length or loss) of an animal (such as a cow), such as one or more modifications described in U.S. Patent Application Publication 20110023158, particularly one or more of the modifications described in paragraphs

[0060] -

[0074] , and / or one or more modifications described in U.S. Patent 10,716,298, particularly in columns 3, rows 58-67 (3:58-67), 4:1-67, 5:1-67, 7:1-67; 8:1-18; and / or Table 1, Figure 1 One or more modifications as described herein. U.S. Patent 10,779,518 provides several exemplary genetic markers of fur characteristics, particularly those related to prolactin receptors and genes. In some embodiments, one or more modifications may include one or more modifications such that the modified polynucleotide contains, or does not contain, the genetic markers, SNPs, modifications, or other variant polynucleotides described in U.S. Patent 10,779,518 where the marker indicates an undesirable characteristic, particularly in 1:34-54, 18:16-44 and 63-67, 19:1-8, Figure 1 Genetic markers, SNPs, modifications, or other variant polynucleotides at position -5.

[0214] In some embodiments, the genome is modified to contain one or more modifications that can affect growth rate, feed efficiency, or other aspects of growth and / or development and energy utilization, such as any one or more of any genes or biomarkers, or including any one or more SNPs or other modifications as described in U.S. Patent Application Publication 20080177597, particularly in

[0013] ,

[0147] ,

[0219] , Tables 1-10, 14-16 and Figure 1-19Modifications at the following locations; any one or more of the genes, markers, SNPs and / or modifications shown in U.S. Patent Application Publication 20020142315, particularly the modifications in paragraphs

[0009] -

[0010] ,

[0013] ,

[0021] ,

[0038] -

[0044] ,

[0095] ,

[0097] -

[0103] , Tables 1-2, or any combination thereof.

[0215] In some embodiments, the genome is modified to contain one or more modifications that affect one or more characteristics associated with animal production and / or management, including but not limited to birth weight, calving difficulty, fertility, reproductive capacity, weaning weight, one-year weight, dry matter intake, etc., and the one or more modifications include, but are not limited to, any one or more modifications of any gene, biomarker, polynucleotide and / or any one or more modifications or variations shown in U.S. Patent Application Publication 20090181386, particularly the modifications in paragraphs

[0011] -

[0030] ,

[0032] -

[0060] ,

[0083] ,

[0088] -

[00237] , Tables 2a-20j, 20k1-20k19; and one or more modifications shown in U.S. Patent Application Publication 20070026404, particularly in paragraphs

[0103] -

[0104] . Modifications at

[0006] , one or more modifications shown in U.S. Patent Application Publication 20060172329, particularly the modifications at paragraphs

[0007] ,

[0009] ,

[0012] ,

[0016] ,

[0077] and Tables 1-2; one or more modifications shown in U.S. Patent Application Publication 20150344974, particularly the modifications at

[0018] -

[0023] ,

[0029] ,

[0080] Modifications at

[0081] ,

[0012] -

[0013] and Tables 3-7; one or more modifications as shown in U.S. Patent 7,879,552, particularly in columns 2: rows 40-67, columns 3: rows 10-24, columns 4: rows 29-34, columns 5: rows 18-21, columns 16: rows 6-37, columns 29: rows 13-41, columns 30: rows 15-41, Tables 1-5 and Figure 3Modifications at A-3D; one or more modifications as shown in U.S. Patent Application Publication 20070065843, particularly modifications at paragraphs

[0097] -

[0100] ,

[0130] ,

[0155] ,

[0164] -

[0165] ,

[0216] -

[0218] , and Tables 2, 4-5; one or more modifications as shown in U.S. Patent Application Publication 20100009374, particularly modifications at paragraphs

[0013] ,

[0036] -

[0039] ,

[0050] -

[0051] ,

[0070] ,

[0077] and Tables 2A-2B, 4-5, 7, 9, 11, 12, or any combination thereof.

[0216] In some embodiments, the genome is modified to contain one or more modifications that can affect temperament, including but not limited to modifications in the PEG3 gene, such as any modifications described in U.S. Patent Application Publication 20030018987, particularly the modification at paragraph

[0004] .

[0217] In some embodiments, the genome is modified to contain one or more modifications that can affect pulmonary artery pressure, including but not limited to modifications of EPAS1 or other related genes, such as those described in U.S. Patent 10,138,522, and / or any of the SNPs or other modifications, particularly in columns 1: rows 58-67, columns 2: rows 1 and 20-61, columns 9: rows 12-15, columns 29: rows 24-36, columns 30: rows 30-34, Tables 1-3, and... Figure 7 -9 is the modification. Modifications for producing bovine bioreactors

[0218] In some embodiments, the genome is modified to contain one or more modifications, such as exogenous and / or heterologous genes or regulatory elements, which may enable an animal (e.g., a bovine) to become a bioreactor capable of producing one or more endogenous or exogenous proteins, lipids, or other biological products, for example, in bodily fluids, which may optionally be collected from the bodily fluids and provided to subjects in need. The use of bovines as bioreactors is known in the art. See, for example, Monzani et al., Adv Exp Med Biol. 2022; 1354:299-314; Keefer et al., Council for Agricultural Science and Technology (CAST). 2007. The Role of Transgenic Livestock in the Treatment of Human Disease. Issue Note 35. CAST, Ames, Iowa (CAST, Ames, Iowa); Colman. American Journal of Clinical Nutrition (Am J. Clin. Nut.) 1996. 63:639S-645S; and Wall et al., Journal of Dairy Science 1997. 80:2213-2224. For example, if an endogenous bovine gene product is desired, the genome can be modified to overexpress the desired endogenous bovine gene product in, for example, milk from an animal. The desired gene product can then be purified from the milk. In other instances, the production of a heterologous protein is desired. In these cases, the donor cell genome can be modified to express the desired heterologous protein (e.g., by inserting a transgene corresponding to the desired heterologous protein). In some cases, this can replace the milk protein coding region, causing the endogenous milk protein promoter to drive the transgene production in mammary tissue. The desired heterologous protein can then be purified from the milk. Other instances will be understood given the description herein. In some embodiments, the exogenous desired heterologous protein or other gene product is a therapeutic protein or other gene product. Gametes, chromosomes, alleles, or gene selection bias

[0219] In some embodiments, genomic modifications introduce one or more modifications that provide genetically mediated selection or bias (or selection against undesirable sperm or oocytes) of intracytoplasmic sperm or oocytes carrying desired genotypes, alleles, chromosomes, etc., such as those modifications shown in U.S. Patent Application Publication 20210324340, engineered genes, etc. (modification of the SRY gene), and one or more modifications shown in U.S. Patent Application Publication 20200399661, particularly in paragraphs

[0034] -

[0051] ,

[0164] ,

[0168] , Figure 1 Modifications at A-9C and Examples 1-12. Other modifications

[0220] In some embodiments, the genome is modified to contain one or more SNPs shown in a bovine SNP database that is publicly available at animalgenome.org / bioinfo / resources / util / q_bovsnp.html.

[0221] In some embodiments, any modification or SNP shown in Casas and Kehrli Jr., Frontiers in Veterinary Science, December 15, 2016, doi.org / 10.3389 / fvets.2016.00113, particularly the modifications or SNPs in Table 1, Ma et al., Agriculture, 2021, 11, 1018, doi.org / 10.3390 / agriculture11101018, particularly the modifications in Tables 1-3, Keogh et al., Animals, Vol. 15, No. 1, January 2021, 100011, particularly the modifications in Tables 4 and 5; Costilla et al., Genetics, Selection and Evolution Any one or more modifications shown in *Selection Evolution*, Vol. 52, Article No. 51 (2020); Dyle et al., *Genetic Selection Evolution*, Vol. 52, Article No. 2 (2020); Shao et al., 2021, *Front Genet.*, 12:617128; Ortega 2018, *Anim Reprod*, Vol. 15, Supplement 1, pp. 923-932, dx.doi.org / 10.21451 / 1984-3143-AR2018-0018; Halli et al., 2021, *PLOS ONE*, 16(10):e0258216, doi.org / 10.1371 / journal.pone.0258216; Thomson et al., *Canadian Journal of Animal Science*, 16(10):e0258216; Journal of Animal Science, 2013, 93(3):295-306, doi.org / 10.4141 / cjas2012-136; Sweett et al., 2020 Scientific Reports, Vol. 10, Article No.: 20102; Hirwa et al., 2011 Asian Journal of Animal Science, 5(1):34-45; Marete et al., 2018 PLOS ONE, 13(7):e0199931, doi.org / 10.1371 / journal.pone.0199931; Paredes-Sanchez et al., 2020, Rev. Mex Cinc Pecu 11(3):894-904, doi.org / 10.22319 / rmcp.v11i3.5279; Lim et al., 2014, International Journal of Genomics (Int. J Genomic) Article ID: 708562, doi.org / 10.1155 / 2014 / 708562; Weikard et al., 2005, Computational Genomics 21(1); Buzanskas et al., 2017, Journal of Animal Science and Biotechnology 8:67; Bouwman et al., 2018, Meta-analysis of genome-wide association of bovine height identified common genes regulating mammalian body size (Meta-analysis of genome-wide association) Studies for cattle stature identify common genes that regulate body size in mammals. Nature Genetics 50:362-367, DOI:10.1038 / s41588-018-0056-5; Ali et al., 2020, Ann. Anim. Sci., Vol. 20, No. 2:409-423; De Leon et al., 2019, Genet. Molecular. Res. 18(3):gmr18373; Sun et al., 2021, Journal of Dairy Research 88(3):247-252; Baqir et al., 2015, Journal of Applied Animal Research (J App.Animal.Res.)》44(1):380-383, doi.org / 10.1080 / 09712119.2015.1091333;Alvarenga et al., 2021, "Animal" 11(3):715.doi.org / 10.3390 / ani11030715;Fang et al., 2020, "Genome Research (Genome Res.)")》30:790-801; Dunner et al., 2013, Livestock Science 154(1-3):34-44; Li et al., 2011, DNA and Cell Biology 30(1):247-254; or any combination thereof. Non-engineered donor cells

[0222] In some embodiments, donor cells derived from donor cell sources are not genetically modified prior to their use in supplementing NANOS3-deficient hosts. In some embodiments, such cells may be derived from a desired breed, lineage, or specific male or female animal. In some embodiments, such unengineered donor cells have or are believed to contain “elite genetics” or are otherwise derived from or obtained from “genetic elite animals.” The phrase “elite genetics” or “genetic elite” is a term in the art that refers to the genetic composition of an animal (such as a cow) or its cells that represents a superior genetic aberration (i.e., top or bottom, depending on the trait, phenotype, genotype, etc., of the contemporary population, such as 0.0001% to 10%, 0.0001% to 0.001%, 0.001% to 0.01%, 0.01% to 0.1%, 0.1% to 1.0%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6%-7%, 7%-8%, 8%-9%, 9%-10%, or any value or range thereof). Generate engineered host and donor cells for germline complementation

[0223] Any suitable genetic modification technique or system can be used to modify engineered host cells (e.g., NANOS3-deficient cells) and / or engineered donor cells. Exemplary systems, techniques, and strategies are described below and elsewhere herein. In view of the description herein, those skilled in the art will understand other suitable systems and methods, and these systems and methods are within the scope of this description. Engineered host cells and / or host animals (i.e., NANOS3-deficient cells and / or animals) can be generated using appropriate techniques for preparing genetically modified organisms (such as cattle). These include, but are not limited to, somatic cell nuclear transfer, genetic modification of various pluripotent, totipotent, or other stem cells, including but not limited to embryonic stem cells, primordial germ cells, primordial germ cell-like cells, spermatogonial or oogonial stem cells, induced pluripotent stem cells, fertilized eggs, blastocysts, blastomeres, etc. Exemplary bovine cells used for genome modification to produce host cells, donor cells, and / or animals (such as cattle) are described, for example, in Bogliotti et al., Proceedings of the National Academy of Sciences, February 27, 2018, 115(9)2090-2095 and WO. 2019 / 140260 (Bovine primordial pluripotent embryonic stem cells), Zhao et al., Proceedings of the National Academy of Sciences, April 13, 2021, 118(15)e2018505118; doi.org / 10.1073 / pnas.2018505118 (Bovine extended potential stem cells); Su et al., International Journal of Molecular Sciences (Int.J.Mol.Sci.), 2021, 22(19), 10489; doi.org / 10.3390 / ijms221910489 (Bovine induced pluripotent stem cells); Bressan et al., Stem Cell Research and Therapy (StemCell) Res. Ther.) 2020.11:247 (Bovine induced pluripotent stem cells); Pillai et al., Biology Open, October 15, 2021; 10(10):bio058756.doi:10.1242 / bio.058756 (Bovine induced pluripotent stem cells); Kawaguchi et al., 2015. PLOS ONE 10(8):e0135403.doi.org / 10.1371 / journa l.pone.0135403 (primordial bovine induced pluripotent stem cells); Yuan. Proceedings of the National Academy of Sciences of the United States of America, February 2018, 27:115(9):1962-1962 (bovine pluripotent embryonic cells); Wei et al., Cell Stem Cell, March 4, 2021; 28(3):550-567.e12.doi:10.1016 / j.stem.2020.11.003 (intermediate pluripotent stem cells); Owen et al., 12; 22(1):118.doi:10.1186 / s12864-021-07418-3 (Bovine zygote); Soto et al., 2021. Sci Rep. 11:11045. (Bovine embryonic stem cells); Linher et al., 2009. PLOS ONE 4(12):e8263.doi.org / 10.1371 / journal.pone.0008263 (Bovine primordial germ cell-like cells); Souza et al., Reproduction of domesticated animals (Reprod. Domest. Anim.)》2017;52(2):243-250 (bovine ovarian stem cells); Kim et al., Reproduction, Fertility and Development (Reprod. Fertil. Dev.) 2016,28,1762-1780 (embryonic pluripotent cells); Van Stekelenburg-Hamers et al., Molecular Reproduction and Development (Mol. Reprod. Dev.) 1995,40:444-454 (inner cell mass pluripotent cells); Mitalipova et al., Cloning. 2001,3,59-67 (bovine embryonic pluripotent cells); Stice et al., Biology of Reproduction (Biol. Reprod.) 1996,54:100-110 (embryonic pluripotent cells); Lim et al., Veterinary Obstetrics (Therio.) 2011,76:133-142 (bovine embryonic pluripotent cells); Wu et al. Human, *Scientific Reports* 2016, 6, 1-12 (Bovine Embryonic Pluripotent Cells); Park et al., *Anim. Reprod. Sci.* 2015 (Bovine Pluripotent Cells); Saito et al., *Biology* 1992, 201, 134-141; Iwasaki et al., *Reproductive Biology* 2000, 62, 470-475; Jin, *Cytotechnology* 2012, 64, 379-389 (Bovine Blastocyst-Derived Pluripotent Cells); Huang et al., *PLOS ONE* 2011, 6, e24501 (Bovine Induced Pluripotent Cells); Sumer et al., *Journal of Animal Science* 2011, 89, 2708-2716 (Bovine Induced Pluripotent Cells); Xiang et al., *Journal of the European Federation of Biochemistry and Biochemistry* (FEBS) J.) 2021, 1-18 (Bovine extended potential stem cells); Han et al., Cell Research 2011, 21, 1509-1512 (Bovine induced pluripotent stem cells); Cao et al., International Journal of Biological Sciences (Int. J. Biol. Sci.) 2021, 1-18 (Bovine extended potential stem cells);)》2012,8,498-511 (bovine iPSC); Wang et al., Cell Death and Disease, 2013.4 (e907) (bovine testicular iPSC); Talluri et al., Cell Reprogramming, 2015.17,131-140 (bovine iPSC); Heo et al., Stem Cells Dev., 2015.24,393-402 (bovine iPSC); Malaver-Ortega et al., Stem Cells Int., 2016,1-11 (bovine iPSC); Zhao et al., Tissue Cell, 2017.49,521-527. (single-cell-derived bovine iPSC); Kawaguchi et al., PLOS ONE, 2015 10,e0135403. (Bovine iPSC); Furusawa et al., Reproductive Biology 2013.89:2(1):1-12 (Bovine inner cell mass-derived cells); Aponte et al., Reprod. 2008. 136(5):543-547 (Bovine spermatogonial stem cells); Tajik et al., Iranian Journal of Veterinary Research (Iran J Vet Res.) 2017.18(2):113-118 (Bovine spermatogonial stem cells); Zheng et al., Reproduction 2014.147(3). doi.org / 10.1530 / REP-13-0466 (Bovine spermatogonial stem cells); Oatley et al., 2004. Biological Reports 71(3):942-947 (Bovine spermatogonial stem cells); Lei et al., 2017. Journal of Integr. Agri. 16(11):2547-2557 (Bovine male germline stem cells); McLean, Z., B.Oback and G.Laible. 2020. Embryo-mediated genome editing for accelerated genetic improvement of livestock. Frontiers of Agricultural Science and Engineering Engineering) 7(2):148-160 (Bovine stem cells); Giassetti et al., 2019.Spermatogonial Stem Cell Transplantation: Insights and Outlook for Domestic Animals. Annual Review of Animal Biosciences 7(1):385-401 (SSC); Ciccarelli et al., 2020. Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males. Proceedings of the National Academy of Sciences 117(39):24195-24204 (SSC); Kinoshita et al., 2021. Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species. species). Development 148, dev199901 (blastodiscal stem cells); Zhi et al., 2022, Cell Research. 32:383-400 (ectoderm stem cells); Yu et al., 2021 (intermediate or embryonic PSCs); Xiang et al., 2021. LCDM medium supports the derivation of bovine extended pluripotent stem cells with embryonic and extraembryonic potency in bovine-mouse chimeras from iPSCs and bovine fetal fibroblasts.*Journal of the European Union of Biochemists and Astronautics* 288:4394-4411; and elsewhere in this paper (see, for example, exemplary techniques for producing genetically modified and cloned cattle, and working examples herein). In cases where the donor cells are essentially gametes or embryos, suitable techniques (such as embryo transfer, in vitro fertilization, etc.) can be used to obtain adult engineered animals.

[0224] Exemplary techniques for producing genetically modified and cloned cattle are described, for example, see, for example, Tan, W. et al., (2016) *Transgenic Research*, June 2016; 25(3):273-287; Yum et al., *Journal of Animal Science and Biotechnol.*, 2018; 9:16; Monzani et al., *Bioengineered*, May-June 2016; 7(3):123-131; Chan et al., *Proceedings of the National Academy of Sciences*, November 24, 1998; 95(24):14028-14033; Laible and Wells. (2006) *Transgenic Cattle Applications: The Transition from Promise to Proof, Biotechnology and Genetic Engineering*. Reviews), 22:1,125-150, DOI:10.1080 / 02648725.2006.10648068; Wall et al., 1997. Journal of Dairy Science 80:2213-2224; Ross and Cibelli. Methods in Molecular Biology 2010; 636:155-77. doi:10.1007 / 978-1-60761-691-7_10; Beyhan Z et al., Developmental Biology 2007. PMID:17359962; Iager AE et al., Cloning Stem Cells 2008. PMID:18419249; Ross PJ et al., Reproduction. 2009. PMID:19074500; Wang K et al., Cloning Stem Cells. 2009. PMID:19196039; Arias ME et al., Biological Research (Biol Res.) 2013. PMID:24510147; Bogliotti YS et al., Journal of Visual Experiments (J Vis Exp.) 2016; Bogliotti YS et al., Proceedings of the National Academy of Sciences of the United States of America 2018; Daigneault BW et al., Scientific Reports 2018; Daigneault BW et al., Biotechniques. November 2018; 65(5):281-283. doi:10.2144 / btn-2018-0051; Goszczynski et al., Reproductive Biology, April 1, 2019; 100(4):885-895. doi:10.1093 / biolre / ioy256; Soto and Ross. Transgenic Research, June 2016; 25(3):289-306. doi:10.1007 / s11248-016-992 9-5; Goszczynski et al., Reproduction of Domestic Animals, October 2019; 54 Supplement 4:22-31. doi:10.1111 / rda.13503; Soto et al., Scientific Reports, May 26, 2021; 11(1):11045. doi:10.1038 / s41598-021-90422-0; Owen et al., BMC Genomics (BMC Genomics, February 12, 2021; 22(1):118. doi:10.1186 / s12864-021-07418-3; Henning et al., Scientific Reports, December 18, 2020; 10(1):22309. doi:10.1038 / s41598-020-78264-8; Camargo et al., Frontiers in Genetics, July 2020. :11:570069;Ferre et al., Animals. May 2020; 14(5):991-1004.doi:10.1017 / S1751731119002775;Navarro et al., Reproduction, Fertility and Development. January 2019; 32(2):11-39.doi:10.1071 / RD19272;Owen et al., Scientific Reports. Sep 29 2020; 10(1):16031.doi:10.1038 / s41598-020-72902-x;Young et al., Nature Biotechnology (Nature Biotechnology) Biotechnol.) Feb 2020; 38(2):225-232. doi:10.1038 / s41587-019-0266-0; Hennig et al., Scientific Reports 2022.8:12:2067; Giassetti et al., 2019. Spermatogonial stem cell transplantation: insights and prospects for domestic animals. Annual Review of Animal Biosciences 7(1):385-401 (SSC); Ciccarelli et al., 2020. Donor spermatogenesis after transplantation of sterile NANOS2 knockout female stem cells. Proceedings of the National Academy of Sciences 117(39):24195-24204 (SSC) and references cited therein, which are incorporated herein by reference in their entirety and may be applied to this disclosure.

[0225] In embodiments, the method includes delivering a genetic modification system and / or other optional exogenous cargo polynucleotides and / or peptides and / or components thereof to one or more cells to be modified. Delivery can occur in vivo, in vitro, ex vivo, or in situ. Exemplary delivery compositions, systems, and techniques will be further described below and elsewhere herein. In some embodiments, the modified cells are bovine cells, such as bovine embryonic stem cells, bovine primordial germ cells, bovine primordial germ-like cells, bovine pluripotent stem cells, bovine totipotent stem cells, bovine oogonial stem cells, bovine oogonial cells, bovine spermatogonial stem cells, bovine spermatogonia, bovine germ cells, bovine zygotes, bovine blastocysts, bovine induced pluripotent stem cells (such as cells reprogrammed from somatic cells).

[0226] In some embodiments, the genetic modification system can be used to introduce exogenous or heterologous genes (such as natural genes from another species or organism). In some embodiments, an exogenous engineered gene construct is introduced into a cell. In some embodiments, an exogenous engineered gene construct is introduced into a cell. In some embodiments, the exogenous engineered gene construct is an engineered gene construct capable of selectively excising, destroying germ cells, or otherwise preventing selected germ cells or germ cell progenitor cells from fertilization. In some embodiments, the genetic modification system can be used to perform gene editing.

[0227] Engineered hosts and donor cells can be used, in particular, for germline complementation approaches, in which engineered donors are introduced into germline-depleted hosts to produce alternative male and female animals that can be used in conventional mating schemes to produce offspring derived from donor cells. Exemplary genetic modification system

[0228] In some embodiments, the genetic modification system includes a programmable nuclease system (e.g., a CRISPR (or CRISPR-Cas) system), a zinc finger nuclease (ZFN) system, TALEN, a wide range of nucleases), an RNAi system, a transposon system, or a combination thereof. Various genetic modification systems, including CRISPR-Cas, ZFN, TALEN, and transposon systems, have been used to modify bovine cells and / or produce modified bovines. See, for example, Owen et al., *BMC Genomics*, Vol. 22, Article No. 118 (2021); Yum SY, Lee SJ, Kim HM, Choi WJ, Park JH, Lee WW et al., Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing. *Scientific Reports*, 2016; 6 (27185); Garrels W, Talluri TR, Apfelbaum R, Carratala YP, Bosch P, Potzsch K et al., One-step multiplex transgenesis via sleeping beauty transposition in cattle. *Scientific Reports*, 2016; 6 (21953); Ding S, Wu X, Li G, Han M, Zhuang Y, Xu Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell. 2005; 122:473-83; Li T, Shuai L, Mao J, Wang X, Wang M, Zhang X et al., Efficient production of fluorescent transgenic rats using the piggyBac transposon.Scientific Reports 2016; 6(33225); Alessio AP, Fili AE, Garrels W, Forcato DO, Olmos Nicotra MF, Liuaudat AC et al., Establishment of cell-based transposon-mediated transgenesis in cattle. Veterinary Obstetrics. 2016; 85:1297-311.e2; Kim S, Saadeldin IM, Choi WJ, Lee SJ, Lee WW, Kim BH et al., Production of transgenic bovine cloned embryos using piggybac transposition. Journal of Veterinary Science (J Vet Med Sci.) 2011; 73:1453-7; Liu et al., Nature Communications (Nature) Communications, Vol. 4, Article No.: 2565 (2013); Sun et al., Scientific Reports, Vol. 8, Article No.: 15430 (2018); Luo et al., 2014. Efficient Generation of Myostatin (MSTN) Biallelic Mutations in Cattle Using Zinc Finger Nucleases. PLOS ONE 9(4):e95225, doi.org / 10.1371 / journal.pone.0095225; US Patent Publication 20110023158; Wang et al., Efficient TALEN-mediated gene knockin at the bovine Y chromosome and generation of a sex-reversal bovine.*Cellular and Molecular Life Sciences*, Vol. 78, pp. 5415-5425 (2021); Moghaddassi et al., (2014) TALEN-Mediated Modification of the Bovine Genome for Large-Scale Production of Human Serum Albumin. *PLOS ONE* 9(2):e89631, doi.org / 10.1371 / journal.pone.008963; and U.S. Patent Publication 20170099813. All these references are incorporated herein by reference as expressed in their entirety and may be applied to this disclosure. These and other suitable genetic modification systems for bovine genetic modification are described in more detail below and in working examples such as those herein. CRISPR-Cas system

[0229] In some embodiments, the NANOS3 gene is modified using a CRISPR-Cas system. Exemplary uses of the CRISPR-Cas system for generating NANOS3-deficient cells and organisms are illustrated in the working embodiments herein. Generally, the CRISPR-Cas or CRISPR system as used herein and in documents such as WO 2014 / 093622 refers to the transcripts and other elements involved in the expression of CRISPR-related (“Cas”) genes or the activation of Cas genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portion tracrRNA), tracr pairing sequences (in the context of endogenous CRISPR systems, encompassing “direct repeat sequences” and partial direct repeat sequences processed by tracrRNA), guide sequences (also referred to as “spacers” in the context of endogenous CRISPR systems), or as used herein, “guide RNA” (e.g., RNA used to guide Cas, such as Cas9, e.g., CRISPR RNA and trans-activating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from CRISPR loci. Generally, CRISPR systems are characterized by elements that promote the formation of the CRISPR complex at the site of the target sequence (also referred to as protospacers in the context of endogenous CRISPR systems). See, for example, Shmakov et al., (2015) “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”, *Molecular Cell*, DOI: dx.doi.org / 10.1016 / j.molcel.2015.10.008. CRISPR-Cas systems can be used to edit one or more nucleotides, remove one or more nucleotides, and / or delete one or more nucleotides.

[0230] Any suitable CRISPR-Cas system can be used in the context of this disclosure to modify NANOS3 polynucleotides in host cells or any target polynucleotides in donor cells. In some embodiments, the CRISPR-Cas system is a Class 2 system. Class 1 system

[0231] In some embodiments, the CRISPR-Cas system is a class 1 CRISPR-Cas system. In some example embodiments, a class 1 system may be a type I, type III, or type IV Cas protein as described below: Makarova et al., “Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants”, Nature Reviews Microbiology, 18:67-81 (February 2020), which is incorporated herein by reference in its entirety, particularly as Figure 1 As described on page 326; Koonin EV, Makarova KS. 2019 Origins and evolution of CRISPR-Cas systems. Philosophical Reports of the Royal Society B (Phil. Trans. R. Soc. B) 374: 20180087, DOI: 10.1098 / rstb.2018.0087, especially in Figure 1 and 2 As described in the description. In some embodiments, a Class 1 CRISPR-Cas system is a type IA, IB, IC, IU, ID, IE, and IF system, a type IV-A system, an IV-B system, and a type III-A, type III-D system, a type III-C system, and a type III-B subtype system. In some embodiments, a Class 1 CRISPR-Cas system is a variant system, such as type IA, IB, IE, IF, and IU variants, which may include variants carried by transposons and plasmids, including the IF subtype version encoded by the large family of Tn7-like transposons and the Tn7-like transposon group encoding a similarly degraded IB subtype system. Peters et al., Proceedings of the National Academy of Sciences 114(35)(2017); DOI:10.1073 / pnas.1709035114; see also Makarova et al., The CRISPR Journal, Vol. 1, n5, Fig. 5. Type 2 systems

[0232] In some embodiments, the CRISPR-Cas system is a class 2 CRISPR-Cas system. The difference between a class 2 system and a class 1 system is that the class 2 system has a single, large, multi-domain effector protein. In some example embodiments, the class 2 system may be a type II, type V, or type VI system, as described in: Makarova et al., “Evolutionary classification of CRISPR-Cas systems: the explosion of class 2 and derived variants”, *Nature Review Microbiology*, 18:67-81 (February 2020), which is incorporated herein by reference. In some embodiments, the CRISPR-Cas system is a type II subtype, such as II-A, II-B, II-C1, or II-C2 systems. In some embodiments, the type II CRISPR-Cas system is the Cas9 system. In some embodiments, the CRISPR-Cas system is a V-type subtype, such as VA, V-B1, V-B2, VC, VD, VE, V-F1, V-F1 (V-U3), V-F2, V-F3, VG, VH, VI, VK (V-U5), V-U1, V-U2, or V-U4 systems. In some embodiments, the V-type CRISPR-Cas system includes Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas14, and / or In some embodiments, the CRISPR-Cas system is a type VI subtype, such as VI-A, VI-B1, VI-B2, VI-C, or VI-D. In some embodiments, a type VI CRISPR-Cas system includes Cas13a (C2c2), Cas13b (group 29 / 30), Cas13c, and / or Cas13d. Guide RNA

[0233] The CRISPR-Cas system described herein comprises one or more guide RNAs (which may also be referred to herein as “guide molecules,” “guide polynucleotides,” and “guide sequences”). The terms guide molecule, guide sequence, and guide polynucleotide refer to polynucleotides capable of guiding Cas to a target genomic locus and are used interchangeably in the aforementioned cited documents, such as International Patent Publication No. WO 2014 / 093622. Typically, a guide sequence is any polynucleotide sequence that is sufficiently complementary to the target polynucleotide sequence to hybridize with the target sequence and guide the CRISPR complex to sequence-specific binding to the target sequence. Guide molecules can be polynucleotides. The ability of a guide sequence (within a nucleic acid-targeting guide RNA) to guide the nucleic acid-targeting complex to sequence-specific binding to a target nucleic acid sequence can be assessed by any suitable assay. For example, components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex (including the guide sequence to be tested) can be provided to host cells with the corresponding target nucleic acid sequence, such as by transfection with a vector encoding the components of the nucleic acid-targeting complex, followed by assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by a Surveyor assay (Qui et al., 2004. Biotechnology. 36(4) 702-707). Similarly, the cleavage of the target nucleic acid sequence can be assessed in vitro by providing the target nucleic acid sequence, components of the nucleic acid-targeting complex (including the guide sequence to be tested), and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between the reactions of the test and control guide sequences. Other assays are possible and will be apparent to those skilled in the art.

[0234] The guide molecule can be any polynucleotide sequence that is sufficiently complementary to the target nucleic acid sequence to hybridize with it and guide the nucleic acid targeting complex to bind specifically to the target nucleic acid sequence. In some embodiments, the degree of complementarity is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimal alignment is performed using a suitable alignment algorithm. The optimal alignment can be determined by using any suitable algorithm for aligning sequences. Non-limiting examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., Burrows-Wheeler Aligner), ClustalW, ClustalX, BLAT, Novoalign (Novocraft Technologies, available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0235] A guide sequence can be selected, and thus a nucleic acid targeting guide can be selected to target any target nucleic acid sequence. Target sequences are discussed further below.

[0236] In some embodiments, a nucleic acid targeting guide is selected to reduce the degree of secondary structure within the nucleic acid targeting guide. In some embodiments, when optimal folding occurs, about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer nucleotides in the nucleic acid targeting guide are involved in self-complementary base pairing. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some procedures are based on calculating the minimum Gibbs free energy. An example of such an algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example of a folding algorithm is RNAfold, an online web server developed by the Institute of Theoretical Chemistry at the University of Vienna, which uses a centroid structure prediction algorithm (see, for example, ARGruber et al., 2008, Cell 106(1):23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12):1151-62).

[0237] In some embodiments, the guide RNA or crRNA may comprise, consist substantially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence. In some embodiments, the guide RNA or crRNA may comprise, consist substantially of, or consist of a direct repeat sequence fused to or linked with the guide sequence or spacer sequence. In some embodiments, the direct repeat sequence may be located upstream of the guide sequence or spacer sequence (i.e., 5'). In other embodiments, the direct repeat sequence may be located downstream of the guide sequence or spacer sequence (i.e., 3').

[0238] In some embodiments, the crRNA comprises a stem loop, preferably a single stem loop. In some embodiments, the direct repeat sequence forms a stem loop, preferably a single stem loop.

[0239] In some embodiments, the spacer length of the guide RNA is 15 to 35 nucleotides. In some embodiments, the spacer length of the guide RNA is at least 15 nucleotides. In some embodiments, the spacer length is 15 to 17 nucleotides, such as 15 nucleotides, 16 nucleotides, or 17 nucleotides; 17 to 20 nucleotides, such as 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides; 20 to 24 nucleotides, such as 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, or 24 nucleotides; 23 to 25 nucleotides, such as 23 nucleotides, 24 nucleotides, or 25 nucleotides; 24 to 27 nucleotides, such as 24 nucleotides, 25 nucleotides, 26 nucleotides, or 27 nucleotides; 27 to 30 nucleotides, such as 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides; 30 to 35 nucleotides, such as 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, or 35 nucleotides, or 35 nucleotides or longer.

[0240] The term "tracrRNA" or similar terms includes any polynucleotide sequence that is sufficiently complementary to a crRNA sequence for hybridization. In some embodiments, when optimally aligned, the complementarity of the tracrRNA and crRNA sequences along the shorter of the two sequences is about or greater than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the length of the tracr sequence is about or greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides. In some embodiments, the tracr and crRNA sequences are contained within a single transcript such that hybridization between them produces a transcript with a secondary structure (such as a hairpin).

[0241] Generally, complementarity refers to the optimal alignment of the SCA and TRACR sequences along the shorter of the two sequences. Optimal alignment can be determined by any suitable alignment algorithm and can further illustrate secondary structures, such as self-complementarity within the SCA or TRACR sequences. In some embodiments, when optimally aligned, the complementarity of the TRACR and SCA sequences along the shorter of the two sequences is approximately or greater than approximately 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.

[0242] In some embodiments, the complementarity between the guide sequence and its corresponding target sequence can be approximately or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%; the length of the guide RNA or sgRNA can be approximately or greater than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides; or the length of the guide RNA or sgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides; and tracr The RNA can be 30 or 50 nucleotides long. In some embodiments, the complementarity between the guide sequence and its corresponding target sequence is greater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or 99% or 99.5% or 99.9% or 100%. Off-target is defined as the complementarity between the sequence and the guide being less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80%, while off-target is defined as the complementarity between the sequence and the guide being 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5%.

[0243] In some embodiments, the guide RNA (capable of guiding Cas to the target locus) may include (1) a guide sequence capable of hybridizing with a genomic target locus in a eukaryotic cell; (2) a tracr sequence; and (3) a tracr pairing sequence. All (1) to (3) may reside in a single RNA, i.e., an sgRNA (arranged with a 5' to 3' orientation), or the tracr RNA may be a different RNA from the RNA containing the guide and tracr sequences. The tracr hybridizes with the tracr pairing sequence and guides the CRISPR / Cas complex to the target sequence. When the tracr RNA is located on an RNA different from the RNA containing the guide and tracr sequences, the length of each RNA may be optimized to shorten it from its corresponding natural length, and each may be independently chemically modified to prevent degradation by cellular RNases or otherwise increase stability.

[0244] Many modifications to the guide sequence are known in the art and are within the spirit and scope of this disclosure. Various modifications can be used to increase the specificity of binding to the target sequence and / or increase the activity of the Cas protein and / or reduce off-target effects. Example guide sequence modifications are described in particular in paragraphs

[0178] -

[0333] of International Patent Application WO2020033601. The International Patent Application is incorporated herein by reference. Target sequence, PAM and PFS

[0245] In the context of CRISPR complex formation, a "target sequence" refers to a sequence in which the guide sequence is designed to be complementary, wherein hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. The target sequence may comprise an RNA polynucleotide. The term "target RNA" refers to an RNA polynucleotide that is or includes the target sequence. Similarly, as used in the context herein, "target polynucleotide" refers to a polynucleotide sequence that is or includes the target sequence of a guide polynucleotide. In other words, a target polynucleotide may be a polynucleotide or a portion of a polynucleotide designed to be complementary to a portion of the guide sequence, and effector function mediated by a complex comprising a CRISPR effector protein and a guide molecule is directed to the polynucleotide or portion of the polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell.

[0246] The guide sequence can specifically bind to the target sequence in the target polynucleotide. The target polynucleotide can be DNA. The target polynucleotide can be RNA. The target polynucleotide can have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., or more) target sequences. The target polynucleotide can be located on a vector. The target polynucleotide can be genomic DNA. The target polynucleotide can be additional. Other forms of the target polynucleotide are described elsewhere in this document.

[0247] In some embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of: messenger RNA (mRNA), premRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double-stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and small cytoplasmic RNA (scRNA). In some preferred embodiments, the target sequence (also referred to herein as the target polynucleotide) may be a sequence within an RNA molecule selected from the group consisting of mRNA, premRNA, and rRNA. In some preferred embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of ncRNA and lncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a premRNA molecule. PAM and PFS components

[0248] The PAM element is a sequence that can be recognized and bound by the Cas protein. The Cas protein / effect complex can then unwind the dsDNA at a location adjacent to the PAM element. It should be understood that Cas proteins targeting RNA and systems including said Cas proteins do not require a PAM sequence (Marraffini et al., 2010. Nature. 463:568-571). Instead, many substances rely on PFS, which will be discussed elsewhere in this document. In some embodiments, the target sequence should be associated with either a PAM (protospacer adjacent motif) or a PFS (protospacer flanking sequence or site), i.e., a short sequence recognized by the CRISPR complex. Depending on the nature of the CRISPR-Cas protein, the target sequence should be selected such that the complementary sequence of said target sequence in the DNA duplex (also referred to herein as the non-target sequence) is located upstream or downstream of the PAM. In embodiments, the complementary sequence of the target sequence is downstream or 3' of the PAM, or upstream or 5' of the PAM. The precise sequence and length requirements of the PAM vary depending on the Cas protein used, but the PAM is typically a 2-5 base pair sequence adjacent to the protospacer (i.e., the target sequence). Examples of natural PAM sequences for different Cas proteins are provided below, and those skilled in the art will be able to identify additional PAM sequences for use with a given Cas protein.

[0249] The ability to recognize different PAM sequences depends on the Cas peptides included in the system. See, for example, Gleditzsch et al., 2019. RNA Biology. 16(4):504-517. Table 1 below (from Gleditzsch et al., 2019) shows several Cas peptides and the PAM sequences they recognize.

[0250] In a preferred embodiment, the CRISPR effector protein can recognize 3'PAM. In some embodiments, the CRISPR effector protein can recognize 3'PAM as 5'H, where H is A, C, or U.

[0251] Furthermore, engineering of the PAM interaction (PI) domain on Cas proteins can allow for PAM-specific programming, improve target site recognition fidelity, and increase the versatility of CRISPR-Cas proteins, as described by Kleinstiver BP et al. for Cas9. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. July 23, 2015; 523(7561):481-5. doi:10.1038 / nature14592. As will be understood by those skilled in the art as further detailed herein, Cas13 proteins can be similarly modified. Gao et al., "Engineered Cpf1 Enzymes with Altered PAM Specificities," BioRxiv 091611; doi:dx.doi.org / 10.1101 / 091611 (December 4, 2016). Doench et al., Nature Biotechnology, December 2014; 32(12):1262-7, created a pool of sgRNAs, plated across all possible target sites of a small group of six endogenous mouse and three endogenous human genes, and quantitatively assessed their ability to generate null alleles of their target genes by antibody staining and flow cytometry. Doench et al. demonstrated that optimization of PAM improved activity and also provided online tools for designing sgRNAs. Such methods are applicable to this disclosure.

[0252] PAM sequences can be identified in polynucleotides using appropriate design tools, which are commercially available or available online. Such freely available tools include, but are not limited to, CRISPRFinder and CRISPRTarget. (References: Mojica et al., 2009. *Microbiology* 155(Pt.3):733-740; Atschul et al., 1990. *Journal of Molecular Biology* 215:403-410; Biswass et al., 2013. *RNA Biology* 10:817-827; and Grissa et al., 2007. *Nucleic Acid Research* 35:W52-57.) Experimental methods for PAM identification may include, but are not limited to, plasmid depletion assays (Jiang et al., 2013. Nature Biotechnology 31:233-239; Esvelt et al., 2013. Nature Methods 10:1116-1121; Kleinstiver et al., 2015. Nature 523:481-485), screening using a high-throughput in vivo model called PAM-SCNAR (Pattanayak et al., 2013. Nature Biotechnology 31:839-843 and Leeany et al., 2016. Molecular Cell 16:253), and negative screening (Zetsche et al., 2015. Cell 163:759-771).

[0253] As previously mentioned, RNA-targeting CRISPR-Cas systems are generally independent of PAM sequences. Instead, such systems typically recognize protospacer flanking sites (PFS) rather than PAMs. Therefore, type VI CRISPR-Cas systems typically recognize protospacer flanking sites (PFS) rather than PAMs. PFS stands for PAM analogues targeting RNA targets. Type VI CRISPR-Cas systems employ Cas13. Some Cas13 proteins analyzed to date, such as Cas13a (C2c2) identified from *Leptotrichia shahii* (LShCAs13a), exhibit specific discrimination against the G at the 3' end of the target RNA. The presence of C at the corresponding crRNA repeat site indicates that nucleotide pairing at this position is rejected. However, some Cas13 proteins (e.g., LwaCAs13a and PspCas13b) do not appear to have a PFS preference. See, for example, Gleditzsch et al., 2019. *RNA Biology*. 16(4):504-517.

[0254] Some type VI proteins, such as subtype B, have 5'-recognition of D (G, T, A) and 3'-motif requirements of NAN or NNA. One example is the Cas13b protein identified in Bergeella zoohelcum (BzCas13b). See, for example, Gleditzsch et al., 2019. RNA Biology. 16(4):504-517.

[0255] In general, the type VI CRISPR-Cas system appears to have fewer restrictive rules for substrate (e.g., target sequence) recognition than those systems targeting DNA (e.g., type V and type II). Nuclear targeting and transport sequences

[0256] For modifications of nuclear-targeted polynucleotides, including but not limited to genomic DNA, one or more components of the CRISPR-Cas system may include one or more sequences or signals for nuclear targeting and / or transport. While these are specifically discussed with reference to the CRISPR-Cas system, such sequences and signals may be applied to other genetic modification systems or components thereof discussed elsewhere in this document.

[0257] Such sequences can facilitate the targeting of one or more components of the composition to intracellular sequences. To improve the targeting of the CRISPR-Cas protein and / or nucleotide deaminase protein or its catalytic domain to the nucleus used in the methods of this disclosure, it may be advantageous to provide one or more nuclear localization sequences (NLS) for one or both of these components.

[0258] In some embodiments, the NLS used in the context of this disclosure is heterologous to proteins. Non-limiting examples of NLS include NLS sequences derived from: NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO:12) or PKKKRKVEAS (SEQ ID NO:13); NLS derived from nucleoplasmic proteins (e.g., nucleoplasmic protein bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO:14)); c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO:15) or RQRRNELKRSP (SEQ ID NO:16); hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:17); and sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:13) from the IBB domain of the input protein-α. NO:18); the sequences of fibroid T protein VSRKRPRP (SEQ ID NO:19) and PPKKARED (SEQ ID NO:20); the sequence of human p53 PQPKKKPL (SEQ ID NO:21); the sequence of mouse c-abl IV SALIKKKKKMAP (SEQ ID NO:22); the sequences of influenza virus NS1 DRLRR (SEQ ID NO:23) and PKQKKRK (SEQ ID NO:24); the sequence of hepatitis virus delta antigen RKLKKKIKKL (SEQ ID NO:25); the sequence of mouse Mx1 protein REKKKFLKRR (SEQ ID NO:26); the sequence of human poly(ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:27); and the sequence of steroid hormone receptor (human) glucocorticoid RKCLQAGMNLEARKTKK (SEQ ID NO:28). Generally, one or more NLSs possess sufficient strength to drive detectable accumulation of DNA-targeting Cas proteins in the nucleus of eukaryotic cells. The strength of nuclear localization activity can generally be derived from the number of NLSs in the CRISPR-Cas protein, the specific NLS used, or a combination of these factors. Accumulation in the nucleus can be detected using any suitable technique. For example, detectable markers can be fused to nucleic acid-targeting proteins, allowing the intracellular location to be visualized, such as in combination with devices for detecting nuclear location (e.g., nucleus-specific staining, such as DAPI).The nucleus can also be isolated from the cell, and its contents can then be analyzed by any suitable method for protein detection, such as immunohistochemistry, Western blotting, or enzyme activity assays. Accumulation in the nucleus can also be determined indirectly, such as by assays of the effect on the formation of nucleic acid targeting complexes at target sequences (e.g., assays of deaminase activity), or by assays of altered gene expression activity affected by the formation of DNA targeting complexes and / or DNA targeting.

[0259] CRISPR-Cas and / or nucleotide deaminase proteins may have one or more, such as two, three, four, five, six, seven, eight, nine, ten or more heterologous NLS. In some embodiments, the protein contains about one or more NLS at or near the amino terminus, and about one or more NLS at or near the carboxyl terminus, or combinations thereof (e.g., containing zero or at least one or more NLS at the amino terminus and zero or more NLS at the carboxyl terminus). When more than one NLS is present, each NLS may be selected independently of the other NLS, such that a single NLS may exist in more than one copy and / or in combination with one or more other NLS present in one or more copies. In some embodiments, an NLS is considered to be close to the N-terminus or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N-terminus or C-terminus. In a preferred embodiment of the CRISPR-Cas protein, the NLS is attached to the C-terminus of the protein.

[0260] In a CRISPR-Cas system including a deaminase, the CRISPR-Cas protein and the deaminase protein are delivered to cells or expressed intracellularly as separate proteins. In these embodiments, each of the CRISPR-Cas and the deaminase protein may have one or more NLS as described herein. In some embodiments, the CRISPR-Cas and the deaminase protein are delivered to cells or expressed as fusion proteins with cells. In these embodiments, one or both of the CRISPR-Cas and the deaminase protein have one or more NLS. When the nucleotide deaminase is fused with an adaptor protein (such as MS2) as described above, one or more NLS may be provided on the adaptor protein, provided that this does not interfere with aptamer binding. In certain embodiments, one or more NLS sequences may also serve as linker sequences between the nucleotide deaminase and the CRISPR-Cas protein.

[0261] In some embodiments, the components of the CRISPR-Cas system include one or more nuclear export signals (NES), one or more nuclear localization signals (NLS), or any combination thereof. In some cases, the NES may be HIV RevNES. In some cases, the NES may be MAPK NES. When the component is a protein, the NES or NLS may be located at the C-terminus of the component. In some embodiments, the NES or NLS may be located at the N-terminus of the component. In some instances, the Cas protein and optionally the nucleotide deaminase protein or its catalytic domain contain one or more heterologous nuclear export signals (NES) or nuclear localization signals (NLS), preferably HIV Rev NES or MAPK NES, preferably C-terminus. Donor template

[0262] In some embodiments, the CRISPR-Cas system includes a donor nucleic acid, such as a donor template, for example a recombinant template, as discussed elsewhere in this disclosure. The template may be a component of another vector as described herein, contained within a separate vector, or provided as a separate polynucleotide. In some embodiments, the recombinant template is designed to act as a template in homologous recombination, such as within or near a target sequence cleaved or cut by a nucleic acid targeting effector protein as part of a nucleic acid targeting complex.

[0263] In one embodiment, the template nucleic acid alters the sequence at the target site. In another embodiment, the template nucleic acid causes the incorporation of modified or non-naturally occurring bases into the target nucleic acid.

[0264] The template sequence can undergo break-mediated or catalytic recombination with the target sequence. In one embodiment, the template nucleic acid may include a sequence corresponding to a site on the target sequence cleaved by a Cas protein-mediated cleavage event. In one embodiment, the template nucleic acid may include sequences corresponding to both a first site on the target sequence cleaved in a first Cas protein-mediated event and a second site on the target sequence cleaved in a second Cas protein-mediated event.

[0265] In some embodiments, the template nucleic acid may include sequences that cause changes to the coding sequence of the translated sequence, such as sequences that cause one amino acid to replace another in the protein product, for example, converting a mutant allele to a wild-type allele, converting a wild-type allele to a mutant allele, and / or introducing a stop codon, inserting an amino acid residue, deleting an amino acid residue, or a nonsense mutation. In some embodiments, the template nucleic acid may include sequences that cause changes to the non-coding sequence, such as changes to exons or 5' or 3' untranslated or untranscribed regions. Such changes include changes to control elements (e.g., promoters, enhancers) and changes to cis- or trans-acting control elements.

[0266] Template nucleic acids homologous to the target site in the target gene can be used to alter the structure of the target sequence. The template sequence can be used to alter unwanted structures, such as unwanted or mutant nucleotides. The template nucleic acid may include sequences that, upon integration, result in: decreased activity of a positive control element; increased activity of a positive control element; decreased activity of a negative control element; increased activity of a negative control element; decreased gene expression; increased gene expression; increased resistance to a symptom or disease; increased resistance to viral entry; correcting mutations or altering unwanted amino acid residues, thereby conferring, increasing, eliminating, or reducing biological properties of the gene product, such as increasing the enzymatic activity of an enzyme or increasing the ability of the gene product to interact with another molecule.

[0267] Template nucleic acids may include sequences that cause sequence changes of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides of the target sequence.

[0268] The template polynucleotide can have any suitable length, such as about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000 or more nucleotides. In one embodiment, the length of the template nucleic acid can be 20+ / -10, 30+ / -10, 40+ / -10, 50+ / -10, 60+ / -10, 70+ / -10, 80+ / -10, 90+ / -10, 100+ / -10, 110+ / -10, 120+ / -10, 130+ / -10, 140+ / -10, 150+ / -10, 160+ / -10, 170+ / -10, 180+ / -10, 190+ / -10, 200+ / -10, or 210+ / -10 nucleotides out of 220+ / -10 nucleotides. In one embodiment, the length of the template nucleic acid can be 30+ / -20, 40+ / -20, 50+ / -20, 60+ / -20, 70+ / -20, 80+ / -20, 90+ / -20, 100+ / -20, 110+ / -20, 120+ / -20, 130+ / -20, 140+ / -20, 150+ / -20, 160+ / -20, 170+ / -20, 180+ / -20, 190+ / -20, 200+ / -20, or 210+ / -20 nucleotides out of 220+ / -20 nucleotides. In one embodiment, the template nucleic acid is 10 to 1,000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, or 50 to 100 nucleotides in length.

[0269] In some embodiments, the template polynucleotide is complementary to a portion of the polynucleotide containing the target sequence. During optimal alignment, the template polynucleotide may overlap with one or more nucleotides of the target sequence (e.g., about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more nucleotides). In some embodiments, during optimal alignment of the template sequence and the polynucleotide containing the target sequence, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides of the target sequence.

[0270] The exogenous polynucleotide template contains the sequence to be integrated (e.g., a mutated gene). The sequence for integration can be endogenous or exogenous within the cell. Examples of sequences to be integrated include polynucleotides encoding proteins or non-coding RNAs (e.g., microRNAs). Therefore, the sequence for integration can be operatively linked to one or more suitable control sequences. Alternatively, the sequence to be integrated can provide regulatory functions.

[0271] The upstream or downstream sequence can contain approximately 20 bp to approximately 2500 bp, for example, approximately 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, 1500 bp, 1600 bp, 1700 bp, 1800 bp, 1900 bp, 2000 bp, 2100 bp, 2200 bp, 2300 bp, 2400 bp, or 2500 bp. In some methods, exemplary upstream or downstream sequences have approximately 200 bp to approximately 2000 bp, approximately 600 bp to approximately 1000 bp, or more specifically, approximately 700 bp to approximately 1000 bp.

[0272] The upstream or downstream sequence can contain approximately 20 bp to approximately 2500 bp, for example, approximately 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, 1500 bp, 1600 bp, 1700 bp, 1800 bp, 1900 bp, 2000 bp, 2100 bp, 2200 bp, 2300 bp, 2400 bp, or 2500 bp. In some methods, exemplary upstream or downstream sequences have approximately 200 bp to approximately 2000 bp, approximately 600 bp to approximately 1000 bp, or more specifically, approximately 700 bp to approximately 1000 bp.

[0273] In some embodiments, one or two homologous arms may be shortened to avoid including certain repeating elements. For example, the 5' homologous arm may be shortened to avoid repeating elements. In other embodiments, the 3' homologous arm may be shortened to avoid repeating elements. In some embodiments, both the 5' and 3' homologous arms may be shortened to avoid including certain repeating elements.

[0274] In some embodiments, the exogenous polynucleotide template may further include a biomarker. Such biomarkers can facilitate screening for targeted integration. Examples of suitable biomarkers include restriction sites, fluorescent proteins, or optional biomarkers. The exogenous polynucleotide templates of this disclosure can be constructed using recombinant technologies (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996).

[0275] In some embodiments, the template nucleic acid used to correct mutations can be designed to be used as a single-stranded oligonucleotide. When using a single-stranded oligonucleotide, the length of the 5' and 3' homologous arms can be in the range of up to about 200 base pairs (bp), for example, the length is at least 25 bp, 50 bp, 75 bp, 100 bp, 125 bp, 150 bp, 175 bp, or 200 bp.

[0276] Suzuki et al. described in vivo genome editing via CRISPR / Cas9-mediated homology-independent targeted integration (2016, Nature 540:144-149). The strategies and techniques of Suzuki et al. are applicable to this disclosure. CAS-based dedicated systems Dead Cas (dCas) system

[0277] In some embodiments, the system is a Cas-based system capable of performing specific functions or activities. For example, the Cas protein may be fused to, operatively coupled to, or otherwise associated with one or more functional domains. In some example embodiments, the Cas protein may be a catalytically death-promoting Cas protein (“dCas”) and / or have nicking enzyme activity. A nicking enzyme is a Cas protein that cleaves only one strand of a double-stranded target. In such embodiments, dCas or nicking enzymes provide sequence-specific targeting functionality to deliver functional domains to or near the target sequence. Example functional domains that can be fused to, operably coupled to, or otherwise associated with Cas proteins may be, or include, nuclear localization signal (NLS) domains, nuclear output signal (NES) domains, translation activation domains, transcription activation domains (e.g., VP64, p65, MyoD1, HSF1, RTA, and SET7 / 9), translation initiation domains, translation repression domains (e.g., KRAB domains, NuE domains, NcoR domains, and SID domains, such as the SID4X domain), nuclease domains (e.g., FokI), histone modification domains (e.g., histone acetyltransferases), lightly inducible / controllable domains, chemically inducible / controllable domains, transposase domains, homologous recombination mechanism domains, recombinase domains, and integrase domains, and combinations thereof. Methods for producing Cas9 or nickases for catalytic death, Cas9 (WO 2014 / 204725, Ran et al., Cell. 12 Sep 2013; 154(6):1380-1389), Cas12 (Liu et al., Nature Communications, 8, 2095(2017)) and Cas13 (International Patent Publications Nos. WO2019 / 005884 and WO2019 / 060746), are known in the art and are incorporated herein by reference.

[0278] In some embodiments, the functional domain may have one or more of the following activities: methyltransferase activity, demethylase activity, translation activation activity, translation initiation activity, translation repression activity, transcription activation activity, transcription repression activity, translation release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, molecular switch activity, chemical inducibility, photoinducibility, and nucleic acid binding activity. In some embodiments, one or more functional domains may contain an epitope tag or a reporter gene. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP).

[0279] One or more functional domains may be located at, near, and / or close to the terminal of an effector protein (e.g., a Cas protein). In embodiments having two or more functional domains, each of the two functional domains may be located at or near the terminal of an effector protein (e.g., a Cas protein). In some embodiments, such as those in which the functional domains are operatively coupled to an effector protein, one or more functional domains may be linked to the effector protein (e.g., a Cas protein) via suitable adapters (including, but not limited to, GlySer adapters). When more than one functional domain is present, the functional domains may be identical or different. In some embodiments, all functional domains are identical. In some embodiments, all functional domains are different from each other. In some embodiments, at least two functional domains are different from each other. In some embodiments, at least two functional domains are identical to each other.

[0280] Other suitable functional domains can be found, for example, in International Patent Publication No. WO 2019 / 018423, and the functional domains described herein may be applicable. Split-Cas system

[0281] In some embodiments, the CRISPR-Cas system is a split CRISPR-Cas system. See, for example, Zechee et al., 2015. Nature Biotechnology 33(2):139-142 and International Patent Publication WO2019 / 018423, the composition and techniques of which can be used and / or are applicable to this invention. The split CRISPR-Cas protein is described in detail herein and in documents incorporated herein by reference. In some embodiments, each part of the split CRISPR protein is attached to a member of a specific binding pair, and the members of the specific binding pair maintain proximity of the parts of the CRISPR protein when they bind to each other. In some embodiments, each part of the split CRISPR protein associates with an inducible binding pair. An inducible binding pair is a binding pair that can be “opened” or “closed” by a protein or small molecule that binds to both members of the inducible binding pair. In some embodiments, the CRISPR protein may preferably split between domains, maintaining domain integrity. In certain embodiments, the Cas splitting domain (e.g., RuvC and HNH domains in the case of Cas9) can be introduced into the cell simultaneously or sequentially, allowing the splitting Cas domain to process target nucleic acid sequences in algal cells. The reduced size of the splitting Cas compared to wild-type Cas allows for other methods of delivering the system to cells, such as using cell-penetrating peptides as described herein. DNA and RNA base editing systems

[0282] In some embodiments, a base editing system is used to modify the polynucleotides of this disclosure as described elsewhere herein. For example, in some embodiments, a base editing system is used for genome editing. In some embodiments, the Cas protein is linked or fused with a nucleotide deaminase. Therefore, in some embodiments, the Cas-based system may be a base editing system. As used herein, “base editing” generally refers to a process of modifying polynucleotides by means of a CRISPR-Cas-based or Cas-based system, the process of which does not involve the removal of nucleotides for modification. Base editing can transform base pairs at precise locations without producing excessive and undesirable editing byproducts that might occur with conventional CRISPR-Cas systems.

[0283] In some example embodiments, nucleotide deaminases can be DNA base editors used in combination with DNA-binding Cas proteins (such as, but not limited to, type II and type V systems). Two types of DNA base editors are generally known: cytosine base editors (CBEs) and adenine base editors (ABEs). CBEs convert C·G base pairs to T·A base pairs (Komor et al., 2016. Nature. 533:420-424; Nishida et al., 2016. Science. 353; and Li et al., Nature Biotechnology 36:324-327), and ABEs convert A·T base pairs to G·C base pairs. In general, CBEs and ABEs can mediate all four possible conversion mutations (C to T, A to G, T to C, G to A). Rees and Liu. 2018. Nature Reviews Genetics (Nat. Rev. Genet.) 19(12):770-788, particularly Figure 1 b, 2a-2c, 3a-3f and Table 1. In some embodiments, the base editing system includes CBE and / or ABE. In some embodiments, the base editing system can be used to modify the polynucleotides of the invention described elsewhere herein. Rees and Liu. 2018. Nature Reviews Genetics 19(12):770-788. Base editing generally does not require a DNA donor template and / or depends on homology-directed repair. Komor et al., 2016. Nature. 533:420-424; Nishida et al., 2016. Science. 353; and Gaudeli et al., 2017. Nature. 551:464-471. Upon binding to a target locus in DNA, base pairing between the system's guide RNA and the target DNA strand results in the substitution of a small segment of ssDNA in the "R loop". Nishimasu et al., Cell. 156:935-949. The DNA bases in ssDNA bubbles are modified by enzymatic components (such as deaminases). In some systems, the catalytically disabled Cas protein can be a variant, or the modified Cas can have nicking enzyme function and can create nicks in the unedited DNA strand to induce the cell to use the edited strand as a template to repair the unedited strand. Komor et al., 2016. Nature. 533:420-424; Nishida et al., 2016. Science. 353; and Gaudeli et al., 2017. Nature. 551:464-471, which may apply to this disclosure.

[0284] Other exemplary V-type base editing systems are described in International Patent Publications Nos. WO2018 / 213708, WO2018 / 213726, WO2019126709, WO2019126716 and WO2019126762, each of which is incorporated herein by reference and may be applied to this disclosure.

[0285] In some example embodiments, the base editing system may be an RNA base editing system. Like DNA base editors, nucleotide deaminases capable of converting nucleotide bases can be fused to Cas proteins. However, in these embodiments, the Cas protein needs to be able to bind RNA. Example RNA-binding Cas proteins include, but are not limited to, RNA-binding Cas9, such as *Francisella novicida* Cas9 (“FnCas9”) and the class 2 VI Cas system. The nucleotide deaminase may be a cytidine deaminase or an adenosine deaminase, or an adenosine deaminase engineered to have cytidine deaminase activity. In some example embodiments, the RNA base editor may be used to delete or introduce post-translational modification sites in expressed mRNA. Unlike DNA base editors, whose edits are permanent in the modified cells, RNA base editors can provide editing in situations where more fine temporal control may be required, such as in modulating specific immune responses. Example VI RNA-base editing systems are described in Cox et al., 2017. Science 358:1019-1027, International Patent Publications WO 2019 / 005884, WO2019 / 005886, WO2019 / 071048, and WO2019126709, which are incorporated herein by reference and are applicable to this disclosure. An example FnCas9 system applicable to RNA base editing purposes is described in International Patent Publication WO2016 / 106236, which is incorporated herein by reference and is applicable to this disclosure.

[0286] Levy et al., Nature Biomedical Engineering doi.org / 10.1038 / s41441-019-0505-5 (2019), describe an example method for delivering a base editing system, which includes splitting CBE and ABE into reconfigurable halves using a split intron approach. The literature is incorporated herein by reference and may be applied to this disclosure. First Editor System

[0287] In some embodiments, a leader editing system is used to modify the polynucleotides of this disclosure as described elsewhere herein. See, for example, Anzalone et al., 2019. Nature. 576:149-157. For example, in some embodiments, a leader editing system is used for genome editing. Like base editing systems, leader editing systems can target and modify polynucleotides without producing double-strand breaks and do not require a donor template. Additional leader editing systems can support all 12 possible combinations of exchanges. Leader editing can be operated using a “search and replace” approach and can mediate targeted insertions, deletions, all 12 possible inter-base conversions, and combinations thereof. Typically, leader editing systems exemplified as PE1, PE2, and PE3 (ibid.) may include a reverse transcriptase fused to or otherwise coupled or associated with an RNA-programmable nickase and a guide RNA (pegRNA) for leader editing extension to facilitate direct copying of genetic information from the extension on the pegRNA to the target polynucleotide. Embodiments that can be used with the present invention include these and variations thereof. Compared to traditional CRISPR-Cas systems, lead editing offers advantages such as lower off-target activity, fewer byproducts, and higher efficiency, or efficiency comparable to traditional CRISPR-Cas systems.

[0288] In some embodiments, the lead editing guide molecule may specify target polynucleotide information (e.g., sequence) and contain a new polynucleotide cargo replacing the target polynucleotide. To initiate transfer from the guide molecule to the target polynucleotide, the PE system can cleave the target polynucleotide on the target side to expose the 3' hydroxyl group. This can trigger reverse transcription of the editing-coding extension region of the guide molecule (e.g., a lead editing guide molecule or a peg guide molecule), directly into the target site within the target polynucleotide. See, for example, Anzalone et al., 2019. Nature. 576:149-157, particularly... Figure 1 b, 1c, and related and supplementary discussions provide further details.

[0289] In some embodiments, the lead editing system may consist of a Cas peptide with nicking enzyme activity, a reverse transcriptase, and a guide molecule. The Cas peptide may lack nuclease activity. The guide molecule may include a target-binding sequence, a primer-binding sequence, and a template containing the edited polynucleotide sequence. The guide molecule, the Cas peptide, and / or the reverse transcriptase may be coupled together or otherwise associated with each other to form an effector complex and edit the target sequence. In some embodiments, the Cas peptide is a class 2 V Cas peptide. In some embodiments, the Cas peptide is a Cas9 peptide (e.g., a Cas9 nicking enzyme). In some embodiments, the Cas peptide is fused to the reverse transcriptase. In some embodiments, the Cas peptide is linked to the reverse transcriptase.

[0290] In some embodiments, the pilot editing system may be a PE1 system or a variant thereof, a PE2 system or a variant thereof, or a PE3 system (e.g., PE3, PE3b). See, for example, Anzalone et al., 2019. Nature. 576:149-157, particularly on pages 2-3. Figure 2 a, 3a-3f, 4a-4b, Extended data Figure 3 Descriptions are provided at points a-3b and 4.

[0291] The length of a PEG guide molecule can be from approximately 10 to approximately 200 or more nucleotides, such as 10 to / or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 nucleotides. 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 or more nucleotides. The optimization of the peg guide molecule can be accomplished as described in Anzalone et al., 2019. Nature. 576:149-157, especially on page 3. Figure 2Figures a-2b and extended data 5a-c are described.

[0292] In some embodiments, the variant leader editing system is used to modify the polynucleotides of this disclosure. In some embodiments, the variant leader editing system is a system for programmable addition via site-specific targeting elements (PASTEs), as described in Yarnall et al., Nature Biotechnology (2022). https: / / doi.org / 10.1038 / s41587-022-01527-4 CRISPR-associated transposase (CAST) system

[0293] In some embodiments, the polynucleotides of this disclosure described elsewhere herein may be modified using a CRISPR-associated transposase (“CAST”) system. A CAST system may include a catalytically inactive or engineered Cas protein, and further include a transposase (or a subunit thereof) that catalyzes RNA-guided DNA transposition. Such systems are capable of inserting DNA sequences at target sites in DNA molecules without relying on host cell repair mechanisms. CAST systems may be class 1 or class 2 CAST systems. Example class 1 systems are described in Klompe et al., Nature, doi:10.1038 / s41586-019-1323, which is incorporated herein by reference. Example class 2 systems are described in Strecker et al., Science, 10 / 1126 / science.aax9181 (2019) and PCT / US2019 / 066835, which are incorporated herein by reference and may be applied to this disclosure. TALE nuclease

[0294] In some cases, the site-directed nuclease is a TALE polypeptide. In some embodiments, a TALE nuclease or TALE nuclease system can be used to modify polynucleotides, such as the NANOS3 gene or polynucleotides in donor cells in the complementation system described herein. In some embodiments, the methods provided herein use isolated, non-naturally occurring, recombinant, or engineered DNA-binding proteins comprising TALE monomers or TALE monomers or half-monomers as part of their organizational structure, thereby enabling targeted nucleic acid sequences with increased efficiency and expanded specificity.

[0295] Naturally occurring TALE, or “wild-type TALE,” is a nucleic acid-binding protein secreted by many species of Proteobacteria. TALE polypeptides contain a nucleic acid-binding domain consisting of a highly conserved tandem repeat sequence of a monomeric polypeptide, the length of which is primarily 33, 34, or 35 amino acids, and which differ primarily from each other at amino acid positions 12 and 13. In an advantageous embodiment, the nucleic acid is DNA. As used herein, the terms “polypeptide monomer,” “TALE monomer,” or “monomer” will be used to refer to a highly conserved repeating polypeptide sequence within the TALE nucleic acid-binding domain, and the terms “repeated variable diresidue” or “RVD” will be used to refer to the highly variable amino acids at positions 12 and 13 of the polypeptide monomer. As provided throughout the disclosure, the amino acid residues of RVD are described using IUPAC amino acid single-letter codes. The general representation of a TALE monomer contained within a DNA-binding domain is X. 1-11 -(X 12 X 13 )-X 14-33 or X 34 or X 35 The subscript indicates the position of the amino acid, and X represents any amino acid. 12 X 13 Indicating RVD. In some polypeptide monomers, the variable amino acid at position 13 is missing or absent, and in such monomers, RVD consists of a single amino acid. In such cases, RVD can alternatively be represented as X*, where X represents X. 12 And (*) indicates X 13 No. The DNA-binding domain contains several repeating sequences of the TALE monomer, and this can be represented as (X... 1-11 -(X 12 X 13 )-X 14-33 or X 34 or X 35 ) z In one advantageous embodiment, z is at least 5 to 40. In another advantageous embodiment, z is at least 10 to 26.

[0296] TALE monomers can possess nucleotide binding affinity determined by the identity of amino acids in their RVD. For example, a polypeptide monomer with RVD NI preferentially binds to adenine (A), a monomer with RVD NG preferentially binds to thymine (T), a monomer with RVD HD preferentially binds to cytosine (C), and a monomer with RVD NN preferentially binds to both adenosine (A) and guanine (G). In some embodiments, a monomer with RVD IG preferentially binds to T. Therefore, the number and order of repeating sequences in the nucleotide-binding domain of a TALE monomer determine its nucleic acid target specificity. In some embodiments, a monomer with RVD NS can recognize all four base pairs and can bind to A, T, G, or C. The structure and function of TALE are further described, for example, in Moscou et al., Science 326:1501 (2009); Boch et al., Science 326:1509-1512 (2009); and Zhang et al., Nature Biotechnology 29:149-153 (2011).

[0297] The polypeptide used in the method of the present invention may be an isolated, non-naturally occurring, recombinant, or engineered nucleic acid binding protein having a nucleic acid or DNA binding region containing a polypeptide monomer repeat sequence, wherein the polypeptide monomer repeat sequence is designed as a target-specific nucleic acid sequence.

[0298] As described herein, RVDs are polypeptide monomers of HN or NH that preferentially bind to guanine, thereby allowing the generation of TALE polypeptides with high binding specificity to target nucleic acid sequences containing guanine. In some embodiments, polypeptide monomers having RVDs RN, NN, NK, SN, NH, KN, HN, NQ, HH, RG, KH, RH, and SS may preferentially bind to guanine. In some embodiments, polypeptide monomers having RVDs RN, NK, NQ, HH, KH, RH, SS, and SN may preferentially bind to guanine, thereby allowing the generation of TALE polypeptides with high binding specificity to target nucleic acid sequences containing guanine. In some embodiments, polypeptide monomers having RVDs HH, KH, NH, NK, NQ, RH, RN, and SS may preferentially bind to guanine, thereby allowing the generation of TALE polypeptides with high binding specificity to target nucleic acid sequences containing guanine. In some embodiments, RVDs with high binding specificity to guanine are RN, NH, RH, and KH. Furthermore, RVD is a polypeptide monomer of NV that preferentially binds to adenine and guanine. In some embodiments, RVD is a monomer of H*, HA, KA, N*, NA, NC, NS, RA, and S* that binds to adenine, guanine, cytosine, and thymine with comparable affinity.

[0299] The predetermined N-terminal to C-terminal sequence of one or more polypeptide monomers containing a nucleic acid or DNA binding domain determines the corresponding predetermined target nucleic acid sequence to which the polypeptide of the present invention will bind. As used herein, monomers and at least one or more half-monomers are “specifically ordered to target” the genomic locus or gene of interest. In plant genomes, the natural TALE binding site always begins with thymine (T), which can be specified by a hidden signal within the non-repetitive N-terminus of the TALE polypeptide; in some cases, this region may be referred to as repeat sequence 0. In animal genomes, the TALE binding site does not necessarily have to begin with thymine (T), and the polypeptide of the present invention can target DNA sequences beginning with T, A, G, or C. The tandem repeat sequence of the TALE monomer always ends with a half-length repeat sequence or sequence segment that may share identity only with the first 20 amino acids of the repeating full-length TALE monomer, and this half-repetitive sequence may be referred to as a half-monomer. Therefore, the length of the targeted nucleic acid or DNA is equal to the number of full-length monomers plus two.

[0300] In some embodiments, TALE may include N-terminal and / or C-terminal capped regions, which can increase the binding efficiency of the TALE peptide (see, for example, Zhang et al., Nature Biotechnology 29:149-153 (2011)). Such “capped regions” may be located directly at the N-terminus and / or C-terminus of the DNA-binding region of the TALE. Exemplary amino acid sequences of N-terminal and C-terminal capped regions are generally known in the art.

[0301] As used herein, the predetermined “N-terminal” to “C-terminal” orientation of the N-terminal capped region, the DNA-binding domain containing the repeating TALE monomer, and the C-terminal capped region provides a structural basis for the organization of different domains in the d-TALE or polypeptide described herein.

[0302] In some embodiments, the entire N-terminal and / or C-terminal capped region is not required to enhance the binding activity of the DNA-binding region. Therefore, in some embodiments, fragments of the N-terminal and / or C-terminal capped region are included in the TALE peptide described herein.

[0303] In some embodiments, the TALE polypeptide described herein contains an N-terminal capped region fragment comprising at least 10, 20, 30, 40, 50, 54, 60, 70, 80, 87, 90, 94, 100, 102, 110, 117, 120, 130, 140, 147, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, or 270 amino acids. In some embodiments, the amino acids of the N-terminal capped region fragment are located at the C-terminus (proximal to the DNA-binding region) of the N-terminal capped region. As described in Zhang et al., Nature Biotechnology 29:149-153 (2011), the N-terminal capped region fragment containing 240 amino acids at the C-terminus enhances the binding activity equal to that of the full-length capped region, while the fragment containing 147 amino acids at the C-terminus retains more than 80% of the efficiency of the full-length capped region, and the fragment containing 117 amino acids at the C-terminus retains more than 50% of the activity of the full-length capped region.

[0304] In some embodiments, the TALE peptides described herein contain a C-terminal capped region fragment comprising at least 6, 10, 20, 30, 37, 40, 50, 60, 68, 70, 80, 90, 100, 110, 120, 127, 130, 140, 150, 155, 160, 170, or 180 amino acids. In some embodiments, the C-terminal capped region amino acids are located at the N-terminus (proximal to the DNA-binding region) of the C-terminal capped region. In some embodiments, the C-terminal capped region comprises only or at least 68 C-terminal amino acids, which enhances the binding activity equal to that of the full-length capped region. See, for example, Zhang et al., Nature Biotechnology 29:149-153 (2011). In some embodiments, the C-terminal capped region comprises only or at least 20 C-terminal amino acids, which has an efficacy of about 50% or more of that of the full-length capped region. See, for example, Zhang et al., Nature Biotechnology 29:149-153 (2011).

[0305] In some embodiments, the capped region of the TALE polypeptide described herein does not need to have the same sequence as the capped region sequence provided herein. Therefore, in some embodiments, the capped region of the TALE polypeptide described herein has a sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or shares identity with the capped region amino acid sequence provided herein. Sequence identity is related to sequence homology. Homology comparison can be performed visually, or more commonly, by means of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of homology (%) between two or more sequences, and can also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences. In some preferred embodiments, the capped region of the TALE polypeptide described herein has a sequence that is at least 95% identical or shares identity with the capped region amino acid sequence provided herein.

[0306] Sequence homology can be generated using any of the many computer programs known in the art, including but not limited to BLAST or FASTA. Alignment can also be performed using suitable computer programs, such as the GCG Wisconsin Bestfit software package. Once the software has generated the best alignment, it is possible to calculate the homology % (percentage of homology), preferably the sequence identity % (percentage of sequence identity). The software typically includes this as part of the sequence comparison and produces numerical results.

[0307] In some embodiments described herein, the TALE peptide includes a nucleic acid binding domain linked to one or more effector domains. The terms "effector domain" or "regulatory and functional domain" refer to a peptide sequence having activity different from that binding to a nucleic acid sequence recognized by the nucleic acid binding domain. By combining the nucleic acid binding domain with one or more effector domains, the peptides of the present invention can be used to target specific target DNA sequences that bind specifically to the nucleic acid binding domain, mediated by one or more functions or activities mediated by the effector domain.

[0308] In some embodiments of the TALE peptide described herein, the activity mediated by the effector domain is biological activity. For example, in some embodiments, the effector domain is a transcriptional inhibitor (i.e., a repressor domain), such as the mSin interaction domain (SID), the SID4X domain, or a fragment of the Krüppel-associated box (KRAB). In some embodiments, the effector domain is a transcriptional enhancer (i.e., an activation domain), such as the VP16, VP64, or p65 activation domain. In some embodiments, nucleic acid binding is linked to, for example, an effector domain, including but not limited to, transposases, integrases, recombinases, degrading enzymes, invertases, proteases, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetyltransferases, nucleases, transcriptional repressors, transcriptional activators, transcription factor recruitment, nuclear localization signals, or cellular uptake signals.

[0309] In some embodiments, the effector domain is a protein domain exhibiting activities including, but not limited to, transposase activity, integrase activity, recombinase activity, degradase activity, invertase activity, protease activity, DNA methyltransferase activity, DNA demethylase activity, histone acetyltransferase activity, histone deacetyltransferase activity, nuclease activity, nuclear localization signaling activity, transcriptional repressor activity, transcription activator activity, transcription factor recruitment activity, or cellular uptake signaling activity. Other preferred embodiments of the invention may include any combination of the active species described herein.

[0310] Various other TALEN-based systems have been described in the art, and their modifications have been reported regularly; see, for example, Boch, Science 326(5959):1509-12 (2009); Mak et al., Science 335(6069):716-9 (2012); and Moscou et al., Science 326(5959):1501 (2009). The use of TALEN based on the “Golden Gate” platform or cloning protocol has been described by several groups; see, for example, Cermak et al., Nucleic Acid Research 39(12):e82(2011); Li et al., Nucleic Acid Research 39(14):6315-25(2011); Weber et al., PLOS ONE 6(2):e16765(2011); Wang et al., J Genet Genomics 41(6):339-47, e.g., May 17, 2014 (2014); and Cermak T et al., Methods in Molecular Biology 1239:133-59(2015), any of which may be applicable to this disclosure. Zinc finger nucleases

[0311] In some embodiments, the site-directed nuclease is a zinc finger protein. In some embodiments, a zinc finger system is used to modify polynucleotides, such as NANOS3 polynucleotides or donor cell polynucleotides. One type of programmable DNA-binding domain is provided by artificial zinc finger (ZF) technology, which involves arrays of ZF modules to target novel DNA-binding sites in the genome. Each finger module in the ZF array targets three DNA bases. Custom arrays of individual zinc finger domains are assembled into ZF proteins (ZFPs).

[0312] Zinc finger proteins can contain functional domains. The first synthetic zinc finger nuclease (ZFN) was developed by fusing a ZF protein with the catalytic domain of the IIS-type restriction enzyme FokI. (Kim, YG et al., 1994, Chimeric restriction endonuclease, Proceedings of the National Academy of Sciences 91, 883-887; Kim, YG et al., 1996, Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain, Proceedings of the National Academy of Sciences 93, 1156-1160). By using paired ZFN heterodimers, each targeting different nucleotide sequences separated by short spacers, increased cleavage specificity can be obtained while reducing off-target activity. (Doyon, Y et al., 2011, Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. *Nature Methods*, 8, 74-79). ZFPs can also be engineered as transcriptional activators and repressors and have been used to target many genes in various organisms. These, and any other ZFN system, can be used to modify the genome, such as the NANOS3 gene. Exemplary methods for genome editing using ZFNs can be found, for example, in U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, and 7,030. U.S. Patents Nos. 215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, all of which are specifically incorporated herein by reference, and their systems and methods may be applied to this disclosure to generate NANOS3-deficient cells and / or organisms.

[0313] Various ZFN-based systems have been described in this field, with modifications regularly reported, and numerous references describe the rules and parameters for guiding ZFN design; see, for example, Segal et al., Proceedings of the National Academy of Sciences 96(6):2758-63 (1999); Dreier B et al., Journal of Molecular Biology 303(4):489-502 (2000); Liu Q et al., Journal of Biochemistry 277(6):3850-6 (2002); Dreier et al., Journal of Biochemistry 280(42):35588-97 (2005); and Dreier et al., Journal of Biochemistry 276(31):29466-78 (2001). Homing endonucleases

[0314] In some embodiments, the genetic modification system is or includes one or more homing endonucleases. Homing endonucleases (HEs) are sequence-specific endonucleases that have long recognition sequences (14-44 base pairs) and frequently cleave DNA with high specificity at unique sites in the genome. At least six known HE families exist, classified as such by their structure, including GIY-YIG, His-Cis boxes, HNH, PD-(D / E)xK, and Vsr classes derived from a wide range of hosts, including eukaryotes, protozoa, bacteria, archaea, cyanobacteria, and bacteriophages. Like ZFNs and TALENs, HEs can be used as an initial step in genome editing to generate DSBs at target loci. Additionally, some natural and engineered HEs cleave only single strands of DNA, thereby acting as site-specific cleaving enzymes. The large target sequence of HEs and the specificity they provide make them attractive candidates for generating site-specific DSBs.

[0315] Various HE-based systems have been described in the art, and their modifications have been reported regularly; see, for example, Steentoft et al., Glycobiology 24(8):663-80 (2014); Belfort and Bonocora, Methods in Molecular Biology 1123:1-26 (2014); Hafez and Hausner, Genome 55(8):553-69 (2012); and a review of the references cited herein, which may be applied to this disclosure. Large-scale nucleases and hybrid large-scale nucleases

[0316] In some embodiments, the site-directed nuclease is a large-scale nuclease or a hybrid large-scale nuclease. In some embodiments, the large-scale nuclease, hybrid large-scale nuclease, or a system thereof can be used to modify polynucleotides, such as NANOS3 polynucleotides or donor cell polynucleotides. A large-scale nuclease is a deoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence having 12 to 40 base pairs). Exemplary large-scale nucleases and methods for using large-scale nucleases are found in U.S. Patent Nos. 8,163,514, 8,133,697, 8,021,867, 8,119,361, 8,119,381, 8,124,369, and 8,129,134, which are specifically incorporated herein by reference. Such methods can be adapted to produce NANOS3-deficient cells and / or organisms.

[0317] Exemplary hybridization-wide nucleases include, but are not limited to, the MegaTal system and the Tev-mTALEN system, which utilize a fusion of a TALE DNA-binding domain and a catalytically active HE, taking advantage of the tunable DNA binding and specificity of TALE and the cleavage sequence specificity of HE; see, for example, Boissel et al., NAR 42:2591-2601 (2014); Kleinstiver et al., G3 4:1155-65 (2014); and Boissel and Scharenberg, Methods in Molecular Biology 1239:171-96 (2015). Other exemplary hybridization of large-scale nucleases includes, but is not limited to, the MegaTev system, which comprises a fusion of a large-scale nuclease (Mega) with a nuclease domain derived from the GIY-YIG homing endonuclease I-TevI ​​(Tev), wherein the two active sites are located on the DNA substrate approximately 30 bp apart and generate two DSBs with incompatible sticky ends; see, for example, Wolfs et al., NAR 42, 8816-29 (2014). RNAi

[0318] In some embodiments, the genetic modification system is an RNA interference (RNAi) system or agent (e.g., shRNA). As used herein, the term "gene silencing" in relation to the activity of an RNAi molecule or system (e.g., siRNA or miRNA) means, in the absence of miRNA or RNA interference molecules, a reduction in the mRNA level of a target gene in a cell compared to the mRNA level found in the cell, by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100%. In a preferred embodiment, the mRNA level is reduced by at least about 70%, about 80%, about 90%, about 95%, about 99%, or about 100%.

[0319] As used herein, the term “RNAi” refers to any type of interfering RNA system or molecule, including but not limited to siRNAi, shRNAi, endogenous microRNAs, long noncoding RNAs, and artificial microRNAs. For example, it includes sequences previously identified as siRNAs, regardless of the mechanism of upstream or downstream RNA processing (i.e., such sequences can still be incorporated into vectors in the context of flanking sequences described herein, even though siRNAs are believed to have specific in vivo processing methods that lead to mRNA cleavage). The term “RNAi” can include both gene-silencing RNAi molecules and RNAi effector molecules that activate gene expression.

[0320] As used herein, "siRNA" refers to a nucleic acid that forms a double-stranded RNA, which, when present in the same cell as a target gene or expressed in the same cell, has the ability to reduce or inhibit the expression of the gene or target gene. The double-stranded RNA siRNA can be formed from complementary strands. In one embodiment, siRNA refers to a nucleic acid capable of forming a double-stranded siRNA. The sequence of the siRNA may correspond to a full-length target gene or its subsequence. Typically, the length of the siRNA is at least about 15-50 nucleotides (e.g., each complementary sequence of the double-stranded siRNA is about 15-50 nucleotides long, and the length of the double-stranded siRNA is about 15-50 base pairs, preferably about 19-30 base nucleotides, preferably about 20-25 nucleotides, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).

[0321] As used herein, “shRNA” or “small hairpin RNA” (also known as stem-loop) is a type of siRNA. In one embodiment, these shRNAs consist of a short (e.g., about 19 to about 25 nucleotides) antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and a similar sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow it.

[0322] The terms “microRNA” or “miRNA” are used interchangeably herein and refer to endogenous RNAs, some of which are known to regulate the expression of protein-coding genes at the posttranscriptional level. Endogenous microRNAs are small RNAs naturally present in the genome that can regulate the production and utilization of mRNA. The term artificial microRNAs include any type of RNA sequence other than endogenous microRNAs that can regulate the production and utilization of mRNA. MicroRNA sequences have been described in publications such as Lim et al., Genes & Development, 17, pp. 991-1008 (2003), Lim et al., Science, 299, 1540 (2003), Lee and Ambros, Science, 294, 862 (2001), Lau et al., Science, 294, 858-861 (2001), Lagos-Quintana et al., Current Biology, 12, 735-739 (2002), Lagos Quintana et al., Science, 294, 853-857 (2001), and Lagos-Quintana et al., RNA, 9, 175-179 (2003), all of which are incorporated herein by reference. Multiple microRNAs can also be integrated into precursor molecules. In addition, miRNA-like stem loops can be expressed in cells as a medium for delivering artificial miRNAs and short interfering RNAs (siRNAs) with the aim of regulating the expression of endogenous genes through miRNA and / or RNAi pathways.

[0323] As used herein, “double-stranded RNA” or “dsRNA” refers to an RNA molecule containing two strands. Double-stranded molecules include molecules that contain a single RNA molecule that folds itself to form a double-stranded structure. For example, the stem-loop structure of a progenitor molecule derived from a single-stranded miRNA is called a premiRNA (Bartel et al., 2004. Cell 1 16:281-297), which contains a dsRNA molecule.

[0324] RNAi molecules can be delivered either as the final active RNAi molecule or via DNA polynucleotides or vectors that encode the RNAi molecule.

[0325] In some embodiments, the RNAi molecule or system targets a NANOS3 RNA molecule, such as NANOS3 mRNA. In some embodiments, the RNAi molecule or system produces an RNAi molecule that binds to NANOS3 RNA and causes its degradation and / or inhibits the translation of NANOS3 mRNA. In some embodiments, the amount of NANOS3 RNA is reduced below a detectable level and / or the amount of NANOS3 protein is reduced to effectively eliminate the function of NANOS3. In some embodiments, the organism expressing the NANOS3-targeting RNAi system lacks germ cells.

[0326] In some embodiments, an RNAi molecule or system targets one or more RNA molecules in a donor cell to target a gene product of interest, thereby producing a cell or organism with a desired phenotype. Exemplary genes that can be modified in donor cells using the RNAi systems described herein are described in more detail elsewhere in this document. transpose subsystem

[0327] In some embodiments, a transposon system is used to modify NANOS3 polynucleotides in host cells or target polynucleotides in donor cells. Exemplary transposon systems that can be used to modify polynucleotides are described herein, and those skilled in the art will understand these exemplary transposon systems in light of this disclosure. In some embodiments, the transposon system is a class I transposon system polypeptide. In some embodiments, the transposon system is a class II transposon system polypeptide. As used herein, a “transposon” (also referred to as a transposable element) is a polynucleotide sequence capable of moving from one location in the genome to another. Several classes of transposons exist. Transposons include retrotransposons (class I transposons) and DNA transposons (class II transposons). Retrotransposons require transcription of a polynucleotide via movement (or transposition) to transpose the polynucleotide to a new genome or polynucleotide. DNA transposons are polynucleotide transposons that do not require transcription of a polynucleotide via movement (or transposition) to transpose the polynucleotide to a new genome or polynucleotide.

[0328] Suitable class I transposon systems include, but are not limited to, any of the LTR and non-LTR retrotransposon systems. Exemplary class I transposon systems include, but are not limited to, CRE, R2, R4, L1, RTE, Tad, R1, LOA, I, Jockey, and CR1 peptides. See, for example, Proceedings of the National Academy of Sciences, November 21, 2006; 103(47):17602-7; Eickbush TH et al., Integration, Regulation, and Long-Term Stability of R2 Retrotransposons, Microbiol Spectr., April 2015; 3(2):MDNA3-0011-2014.doi:10.1128 / microbiolspec.MDNA3-0011-2014; Han JS, Non-long terminal repeat (non-LTR) retrotransposons: mechanisms, recent developments, and unanswered questions, Mob DNA.) 2010 May 12; 1(1):15. doi:10.1186 / 1759-8753-1-15; Malik HS et al., The age and evolution of non-LTR retrotransposable elements, Molecular Biology and Evolution (Mol Biol Evol.) June 1999; 16(6):793-805, the references are incorporated herein by reference in their entirety.

[0329] Suitable class II transposon systems include, but are not limited to, any of the following transposon systems: Sleeping Beauty transposon system (Tc1 / mariner superfamily) (see, for example, Ivics et al. 1997. Cell. 91(4):501-510), piggyBac (piggyBac superfamily) (see, for example, Li et al. 2013 110(25):E2279-E2287 and Yusa et al. 2011. Proceedings of the National Academy of Sciences. 108(4):1531-1536), Tol2 (superfamily hAT), Frog Prince (Tc1 / mariner superfamily) (see, for example, Miskey et al. 2003 Nucleic Acid Research 31(23):6873-6881) and variants thereof. In some embodiments, the class II transposon system is a DD[E / D] transposon or a transposon polypeptide. In some embodiments, the class II transposon system is Tc1 / mariner, PiggyBac, Frog Prince, Tn3, Tn5, hAT, CACTA, P, mutant, PIF / Harbinger, Transib, or Merlin / IS1016 transposon peptide.

[0330] Suitable class II transposon systems and components that can be used in the context of this invention include, but are not limited to, transposon systems and components described in, for example, the following literature: Han et al., 2013. BMC Genomics. 14:71, doi:10.1186 / 1471-2164-14-71; Lopez and Garcia-Perez. 2010. Curr. Genomics. 11(2):115-128; Wessler. 2006. Proceedings of the National Academy of Sciences. 103(47):176000-17601; Gao et al., 2017. Marine Genomics. Genomics. 34:67-77; Bradic et al. 2014. Mobile DNA. 5(12) doi:10.1186 / 1759-8753-5-12; Li et al. 2013. Proceedings of the National Academy of Sciences. 110(25)E2279-E2287; Kebriaei et al. 2017. Trends in Genetics. Genetics. 33(11):852-870); Miskey et al. 2003. Nucleic Acid Research 31(23):6873-6881; Nicolas et al. 2015. Microbiology Spectrum 3(4) doi:10.1128 / microbiolspec.MDNA3-0060-2014; WS Reznikoff. 1993. AnnuRev. Microbiol. 47:945-963; Rubin et al. 2001. Genetics. 158(3):949-957; Wicker et al. 2003. Plant Physiology. Physiol.) 132(1):52-63; Majumdar and Rio. 2015. Microbiology Spectrum 3(2) doi:10.1128 / microbiolspec.MDNA3-0004-2014; D. Lisch. 2002. Trends in Plant Sci. 7(11):498-504; Sinzelle et al. 2007. Proceedings of the National Academy of Sciences of the United States of America. 105(12):4715-4720; Han et al. 2014; Journal of Genome Biology Biol. Evol.) 6(7):1748-1757; Grzebelus et al. 2006; Molecular Genetics and Genomics (Mol. Genet. Genomics.) 275(5):450-459; Zhang et al. 2004. Genetics. 166(2):971-986; Chen and Li.2008. *Gene*. 408(1-2):51-63; and C. Feschotte. 2004. *Molecular Biology and Evolution*. 21(9):1769-1780. Recombinase system

[0331] In some embodiments, the genetic modification system used to modify the genome is a recombinase system. Typically, recombinases are enzymes that catalyze site-specific recombination events, and recombination systems employ such enzymes to achieve site-specific polynucleotide integration or disruption. Many recombinase systems used for gene knock-in, gene knockout, and other genome or polynucleotide modifications are generally known in the art since their introduction decades ago (see, for example, Sauer, B. Molecular Cell Biology 7(6):2087-2096(1987)) and can be used in the context of this disclosure to introduce one or more components of the transgenes of this disclosure and / or another genetic modification system described herein, and / or generally the genome of the cell or another polynucleotide is known. Exemplary systems include, but are not limited to, Cre-lox and FLP-FRT systems (see, for example, Maizels et al., *J. Immunol.* 2013, 161(1): doi:10.4049 / jimmunol.1301241; Graham et al., *Biotech J.* 2009, 4(1):108-118; Chen et al., *Zoological Journal* 4(5):767-771 (2010); Kalds et al., *Frontiers in Genetics* 2019, doi.org / 10.3389 / fgene.2019.00750; Gurusinghe et al., *J. Cell Biochem.* 2017, 118(5):1201-1215; and Wang et al., *Plant Cell Reports*). The references cited herein are incorporated by way of their whole expression and may be applied to this disclosure. (Rep) (2011) 30:267-285. Delivery of polynucleotides and peptides

[0332] Gene modification systems or their components (including any polynucleotides, vectors, vector systems, etc.) can be delivered to cells or cell populations using any suitable delivery composition, system, or technology. Physical delivery

[0333] In some embodiments, genetic modification systems or components thereof can be introduced into cells via physical delivery methods. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery. Both nucleic acids and proteins can be delivered using such methods. For example, Cas proteins can be prepared, isolated (and, if necessary, refolded and purified) in vitro and introduced into cells via physical delivery methods or techniques. Microinjection

[0334] Direct microinjection of genetically modified systems or their components into cells can achieve high efficiency, such as above 90% or about 100%. In some embodiments, microinjection can be performed by using a microscope and a needle (e.g., 0.5–5.0 μm in diameter) to pierce the cell membrane and deliver the cargo directly to the target site within the cell. Microinjection can be used for both in vitro and ex vivo delivery.

[0335] Microinjection can be performed on plasmids containing coding sequences for Cas or other genetically modified system effector proteins and / or any associated polynucleotides (e.g., guide RNA, mRNA, and / or guide RNA). In some cases, microinjection can be used to i) deliver DNA directly to the cell nucleus, and / or ii) deliver mRNA (e.g., in vitro transcribed) to the cell nucleus or cytoplasm. In certain instances, microinjection can be used to deliver sgRNA directly to the cell nucleus and to deliver mRNA encoding Cas or other effector proteins to the cytoplasm, for example, to facilitate the translation and shuttle of Cas or other effector proteins to the cell nucleus.

[0336] Microinjection can be used to produce genetically modified animals. For example, gene-modifying systems or components thereof can be injected into fertilized eggs, blastomeres, blastocysts, embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, primordial germ cells, primordial germ-like cells, etc., to allow for gene therapy such as germline modification.

[0337] Microinjection and nuclear transfer of bovine zygotes, blastocysts, and other cells have been described and used to produce genetically modified cattle. See, for example, Behboodi et al., Journal of Dairy Science, Nov 1993; 76(11):3392-9; Bogliotti et al., Journal of Visual Experimentation, 2016; (116):54465; Galli et al., Cloning and Stem Cells, Sep 2002, 189-196, doi.org / 10.1089 / 15362300260339476; Krisher et al., Transgenic Research Research), Vol. 3, pp. 226-231 (1994); Yum et al., Scientific Reports, Vol. 6, Article No.: 27185 (2016); Krimpenfort et al., Biotechnology (New York), September 1991; 9(9):844-7. doi:10.1038 / nbt0991-844; Krishner et al., Journal of Dairy Science, 1995, 78:1282-1288; Otero et al., Indian Journal of Science and Technology, Vol. 11(31), DOI:10.17485 / ijst / 2018 / v11i31 / 130839, August 2018; Kubisch et al., Journal of Reproduction and Fertility, 1995. The references cited in Rep. Fert. (104:133-139) are incorporated herein by reference as if they were expressed in their entirety and may be applied to this disclosure. Electroporation

[0338] In some embodiments, electroporation can be used to deliver cargo and / or delivery media. Electroporation uses pulsed high-voltage current to temporarily open nanometer-sized pores within the cell membrane of cells suspended in a buffer solution, allowing components with hydrodynamic diameters of tens of nanometers to flow into the cells. In some cases, electroporation can be used for various cell types and efficiently transfer cargo into cells. Electroporation can be used for both in vitro and ex vivo delivery.

[0339] Electroporation can also be used to deliver cargo into the nucleus of mammalian cells by applying specific voltages and reagents, such as through nuclear transfection. Such methods include those described in the following literature: Wu Y et al. (2015). Cell Research 25:67-79; Ye L et al. (2014). Proceedings of the National Academy of Sciences 111:9591-6; Choi PS, Meyerson M. (2014). Nature Communications 5:3728; Wang J, Quake SR. (2014). Proceedings of the National Academy of Sciences 111:13157-62. Electroporation can also be used for in vivo cargo delivery, for example, using the method described in Zuckermann M et al. (2015). Nature Communications 6:7391.

[0340] Electroporation has been used to deliver exogenous polynucleotides and / or peptides into bovine zygotes. See, for example, Lin and Van Eenennaam. Frontiers in Genetics 2021;12:648482, doi.org / 10.3389 / fgene.2021.648482, particularly at Supplementary Table 1. In some embodiments, the voltage and pulse number for delivering exogenous polynucleotides to bovine cells such as fertilized eggs or blastocysts via electroporation are 10-20 V / mm and 2-6 pulses, 10-20 V / mm and 2-3 pulses, 15-20 V / mm and 2-3 pulses, and 15 V / mm and 6 pulses, see, for example, Tanihara, F., Hirata, M., Morikawa, S., Nguyen, NT, Le, QA, Hirano, T. et al. (2019). The effects of electroporation on viability and quality of in vivo-derived bovine blastocysts. blastocysts). Journal of Reproduction and Development (J. Reprod.Dev.) 65, 475-479. doi:10.1262 / jrd.2019-049; Namula, Z., Wittayarat, M., Hirata, M., Hirano, T., Nguyen, NT, Le, QA et al. (2019). Genome mutation after the introduction of the gene editing by electroporation of Cas9 protein (GEEP) system into bovine putative zygotes. In Vitro Cell and Developmental Biology - Animal (In Vitro) Cell. Dev. An.) 55, 598-603; Miao, D., Giassetti, MI, Ciccarelli, M., Lopez-Biladeau, B., and Oatley, JM (2019). Simplified pipelines for genetic engineering of mammalian embryos by CRISPR-Cas9 electroporation dagger.Reproductive Biology 101, 177-187; Ciccarelli, M., Giassetti, MI, Miao, D., Oatley, MJ, Robbins, C., Lopez-Biladeau, B. et al. (2020). Donor spermatogenesis after transplantation of infertile NANOS2 knockout female stem cells. Proceedings of the National Academy of Sciences 117, 24195-24204; Camargo, LSA, Owen, JR, Van Eenennaam, AL, and Ross, PJ (2020). Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos. Embryos). *Frontiers in Genetics* 11:570069; and Wei, J., Gaynor, P., Cole, S., Brophy, B., Oback, B. and Laible, G. (2018). "Developing the laboratory conditions for bovine zygote-mediated genome editing by electroporation," published in *Proceedings of the World Congress on Genetics Applied to Livestock Production*, which are incorporated herein by reference and are applicable to this invention. Fluid dynamics delivery

[0341] Hydrodynamic delivery can also be used to deliver gene-modifying systems, for example, for in vivo delivery. In some instances, hydrodynamic delivery can be performed by rapidly injecting a large volume (8-10% body weight) solution containing the gene-modifying system into the bloodstream of a subject (e.g., a cow). Because blood is incompressible, the large bolus of fluid can lead to an increase in hydrodynamic pressure, which temporarily enhances permeability to endothelial and parenchymal cells, allowing cargo that normally cannot cross cell membranes to enter the cells. This method can be used to deliver naked DNA plasmids and proteins. The delivered gene-modifying system or component can be enriched in the ovary and / or testis. transfection

[0342] Goods, such as nucleic acids and / or peptides, can be introduced into cells via transfection methods used to introduce nucleic acids into cells. Examples of transfection methods include calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendritic polymer transfection, heat shock transfection, magnetic transfection, lipid transfection, puncture transfection, optical transfection, and proprietary agent-enhanced nucleic acid uptake. Nucleic acids and vectors that can encode genetic modification systems and / or components thereof, as well as vector systems, are described in more detail elsewhere herein. Transfection has been used to deliver nucleic acid constructs into bovine cells. See, for example, Tajik et al., *Iran J Vet Res.*, 2017, 18(2):113-118; Jafarnejad et al., *S African J Anim Sci.*, 48, 1 (2018) DOI:10.4314 / sajas.v48i1.13; Duarte et al., *Anim Biotechnol.*, 30 Dec 2020; 1-11. doi:10.1080 / 10495398.2020.1862137; and Osorio, *Genes*, 30 Aug 2017; 626:200-208, which are incorporated herein by reference as if expressed in their entirety and may be applied to this disclosure. Transduction

[0343] Genetically modified systems and / or their components, such as nucleic acids and / or peptides, can be introduced into cells via transduction of viruses, pseudoviruses, and / or virus-like particles. Methods for packaging genetically modified systems and / or their components into viral particles can be accomplished using any suitable viral vector or vector system. Such viral vectors and vector systems are described in more detail elsewhere herein. As used in this context, “transduction” refers to the process of introducing foreign nucleic acids and / or proteins into cells (prokaryotes or eukaryotes) via viruses, pseudoviruses, and / or virus-like particles. After packaging into viruses, pseudoviruses, and / or virus-like particles, the viral particles can be exposed to cells (e.g., in vitro, ex vivo, or in vivo), whereby the viruses, pseudoviruses, and / or virus-like particles infect the cells and deliver cargoes to the cells via transduction. The viruses, pseudoviruses, and / or virus-like particles can optionally be concentrated before exposure to target cells. In some embodiments, a viral titer of the composition containing viral and / or pseudovirus particles can be obtained, and a specific titer can be used to transduce cells. Viral vectors and systems, as well as the generation of viral (or pseudoviruses and / or virus-like particles) delivery particles, are described in more detail elsewhere in this paper. Viral transduction has been used to deliver exogenous nucleic acid constructs into bovine cells. See, for example, Hoffmann et al., *Biology of Reproduction*, Vol. 71, No. 2, August 1, 2004, pp. 405-409, doi.org / 10.1095 / biolreprod.104.028472; Yu et al., (2014) Expression of Intracellular Interferon-Alpha Confers Antiviral Properties in Transfected Bovine Fetal Fibroblasts and Does Not Affect the Full Development of SCNT Embryos. SCNTEmbryos). PLOS ONE 9(7):e94444, doi.org / 10.1371 / journal.pone.0094444; and Wu et al., Scientific Reports, Vol. 6, Article No.: 28343(2016), which are incorporated herein by reference as if expressed in their entirety and may be applied to this disclosure. Gene gun method

[0344] Genetically modified systems and / or components thereof, such as nucleic acids and / or peptides, can be introduced into cells using gene gun methods or techniques. As used herein, the art term "gene gun" refers to the delivery of nucleic acids to cells via high-speed particle bombardment. In some embodiments, the genetically modified system and / or its components may be attached, associated with, or otherwise coupled to particles, which can then be delivered to cells via a gene gun (see, for example, Liang et al. 2018. *Nat. Protocol.* 13:413-430; Svitashev et al. 2016. *Nat. Communications.* 7:13274; Ortega-Escalante et al. 2019. *Plant. J.* 97:661-672). In some embodiments, the particles may be gold, tungsten, palladium, rhodium, platinum, or iridium particles. Implantable devices

[0345] In some embodiments, the delivery system may include an implantable device incorporating or coated with the genetic modification system and / or components thereof described herein. Various implantable devices are described in the art, including any device, graft, or other composition that can be implanted into a subject such as a cow. Delivery medium

[0346] Polynucleotides and / or peptides of this disclosure, such as genetic modification systems, can be delivered (e.g., to target cells to be modified) via one or more delivery media. Delivery media can deliver goods such as polynucleotides or peptides of this disclosure (e.g., genetic modification systems) into cells, tissues, organs, or organisms (e.g., animals or plants). In some embodiments, delivery media are used to deliver goods such as genetic modification systems or components thereof, or other polynucleotides or peptides of this disclosure, to target bovine cells. Goods may be packaged, transported, or otherwise associated with a delivery media. Delivery media may be selected based on the type of goods to be delivered, and / or delivery may be in vitro and / or in vivo. Examples of delivery media include vectors, viruses (e.g., viral particles, pseudovirus particles, or virus-like particles), non-viral media (e.g., exosomes, liposomes, etc.), and other delivery agents described herein and delivery agents understood by those skilled in the art in light of this disclosure.

[0347] The delivery medium described herein has a maximum size or maximum average size (e.g., diameter or maximum average diameter) of less than 100 micrometers (μm). In some embodiments, the maximum size or maximum average size of the delivery medium is less than 10 μm. In some embodiments, the maximum size or maximum average size of the delivery medium may be less than 2000 nanometers (nm). In some embodiments, the maximum size or maximum average size of the delivery medium may be less than 1000 nanometers (nm). In some embodiments, the maximum size or maximum average size (e.g., diameter or average diameter) of the delivery medium may be less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 150 nm, or less than 100 nm or less than 50 nm. In some embodiments, the maximum size or maximum average size of the delivery medium may be in the range between 25 nm and 200 nm. Particles

[0348] In some embodiments, the delivery medium may be or comprise particles. For example, the delivery medium may be or comprise nanoparticles no larger than 1000 nm (e.g., particles having a maximum size or maximum average size (e.g., diameter or maximum average diameter)). Particles may be provided in various forms, such as solid particles (e.g., metals such as silver, gold, iron, titanium, etc.), nonmetals, lipid-based solids, polymers, suspensions of particles, or combinations thereof. Metallic, dielectric, and semiconductor particles, as well as hybrid structures (e.g., core-shell particles), can be prepared.

[0349] Nanoparticles can also be used to deliver compositions and systems to cells, as described in US20130185823, WO2008042156, and WO2015089419. Generally, "nanoparticle" refers to any particle with a diameter less than 1000 nm. In some embodiments, the maximum size or maximum average size (e.g., diameter or average diameter) of the nanoparticles of the present invention is 500 nm or less. In other embodiments, the maximum size or maximum average size of the nanoparticles of the present invention is in the range of 25 nm to 200 nm. In other embodiments, the maximum size or maximum average size of the nanoparticles of the present invention is 100 nm or less. In other embodiments, the maximum size or maximum average size of the nanoparticles of the present invention is in the range of 35 nm to 60 nm. It should be understood that the references to "particle" or "nanoparticle" herein may be interchangeable where appropriate. If nanoparticles made of semiconductor materials are small enough (typically less than 10 nm) that the electron energy level can be quantized, the nanoparticles may also be labeled as quantum dots. Such nanoparticles are used as drug carriers or imaging agents in biomedical applications and can be suitable for similar purposes in this invention. Semi-solid and soft nanoparticles have been fabricated and are within the scope of this invention. Nanoparticles that are half hydrophilic and half hydrophobic are called Janus particles and are particularly effective for stabilizing emulsions. These particles can self-assemble at the water / oil interface and act as solid surfactants.

[0350] Particle characterization (including, for example, characterizing morphology, size, etc.) is performed using a variety of different techniques. Common techniques include electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visible spectroscopy, dual polarization interferometry, and nuclear magnetic resonance (NMR). Characterization (size measurement) can be performed on natural particles (i.e., pre-loaded) or after loading cargoes (e.g., one or more components of a genetically modified system (e.g., a CRISPR-Cas system or components thereof), and may include additional loads and / or excipients) to provide particles of optimal size for delivery of any in vitro, ex vivo, and / or in vivo application of this disclosure. In some embodiments, particle size (e.g., diameter) characterization is based on measurements using dynamic laser scattering (DLS). See also, for example, U.S. Patent Nos. 8,709,843; 6,007,845; 5,855,913; 5,985,309; 5,543,158; and Dahlman et al., Nature Nanotechnology (2014), doi:10.1038 / nnano.2014.84, the descriptions of which can be applied to the particles of this disclosure, methods for preparing and using said particles, and measurements thereof. Carrier and carrier system

[0351] In some embodiments, the delivery medium is a vector or vector system or particle, such as a virus or virus-like particle produced by such a vector or vector system. Therefore, vectors that may contain one or more of the polynucleotides of the genetic modification systems described herein are also provided herein. In some embodiments, the vector may contain one or more polynucleotides encoding one or more elements of the genetic modification systems described herein. The vector can be used to produce bacteria, fungi, yeast, plant cells, animal cells, and transgenic animals that can express one or more components of the genetic modification systems described herein and are therefore genetically modified or conferred the ability to produce particles (e.g., viruses or virus-like particles) that can be used to deliver the genetic modification systems described herein to cells such as bovine cells.

[0352] Vectors containing one or more of the polynucleotide sequences described herein are within the scope of this disclosure, such as vectors associated with the introduction of NANOS3 modification to generate host cells or the modification of donor cell polynucleotides. One or more of the polynucleotides as part of a genetic modification system may be included in a vector or vector system. Vectors and / or vector systems can be used, for example, to express one or more of the polynucleotides in cells such as producer cells to generate genetic modification systems containing viral particles described elsewhere herein. Other uses of the vectors and vector systems described herein are also within the scope of this disclosure. Generally and throughout this specification, the term “vector” refers to a tool that allows or facilitates the transfer of an entity from one environment to another. In some contexts understood by those skilled in the art, “vector” can refer to the art term for a nucleic acid molecule capable of transporting another nucleic acid already linked to it. A vector can be a replicon, such as a plasmid, bacteriophage, or granule, into which another DNA segment can be inserted to achieve replication of the inserted segment. Generally, a vector is capable of replication when associated with appropriate control elements.

[0353] Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, as by standard molecular cloning techniques. Another type of vector is a viral vector, in which a viral DNA or RNA sequence is present for packaging into a virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also include polynucleotides carried by the virus for transfection into host cells. Some vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell and thereby replicate along with the host genome. Furthermore, some vectors are capable of guiding the expression of genes operatively linked to them. These vectors are referred to as "expression vectors" in this paper. Typically, expression vectors used in recombinant DNA technology are in plasmid form.

[0354] Recombinant expression vectors may consist of nucleic acids (e.g., polynucleotides) suitable for expression in host cells, meaning that the recombinant expression vector includes one or more regulatory elements, which may be selected based on the host cell used for expression and are operatively linked to the nucleic acid sequence to be expressed. In recombinant expression vectors, "operatively linked" and "operationally linked" are used interchangeably herein and mean that the nucleotide sequence of interest (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell) is linked to the regulatory element in a manner that allows the expression of the nucleotide sequence. Advantageous vectors include lentiviruses and adeno-associated viruses, and the type of such vector can also be selected to target specific cell types. These and other embodiments of vectors and vector systems are described elsewhere herein.

[0355] In some embodiments, the vector may be a bicistronic vector. In some embodiments, the bicistronic vector may be used for one or more elements of the genetic modification system described herein. In some embodiments, the expression of elements of the genetic modification system described herein may be driven by the CBh promoter or other ubiquitous promoters. When the element of the genetic modification system is RNA, its expression may be driven by a Pol III promoter such as the U6 promoter. In some embodiments, both are combined. Cell-based vector amplification and expression

[0356] Vectors can be introduced and propagated in prokaryotic or eukaryotic cells. In some embodiments, prokaryotes are used to amplify copies of the vector to be introduced into eukaryotic cells, or as intermediate vectors in the production of the vector to be introduced into eukaryotic cells (e.g., amplifying plasmids as part of a viral vector packaging system). Vectors can be virus-based or non-virus-based. In some embodiments, prokaryotes are used to amplify copies of the vector and express one or more nucleic acids, such as a source of one or more proteins, for delivery to a host cell or host organism.

[0357] Vectors can be designed to express one or more elements (e.g., nucleic acid transcripts, proteins, enzymes, and combinations thereof) of the genetic modification system described herein in a suitable host cell. In some embodiments, a suitable host cell is a prokaryotic cell. Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. In some embodiments, a suitable host cell is a eukaryotic cell. In some embodiments, the host cell is a cell modified by the genetic modification system. In some embodiments, the host cell is a producer cell capable of producing particles (e.g., viral particles, virus-like particles, exosomes, etc.) that can be used to deliver the genetic modification system or its components into the cell.

[0358] In some embodiments, suitable host cells are suitable bacterial cells. Suitable bacterial cells include, but are not limited to, bacterial cells derived from the species *Escherichia coli*. Many suitable *E. coli* strains are known in the field of vector expression. These include, but are not limited to, Pir1, Stbl2, Stbl3, Stbl4, TOP10, XL1 Blue, and XL10 Gold. In some embodiments, host cells are suitable insect cells. Suitable insect cells include cells derived from the fall armyworm. Suitable fall armyworm cell strains include, but are not limited to, Sf9 and Sf21. In some embodiments, host cells are suitable yeast cells. In some embodiments, yeast cells may be derived from *Saccharomyces cerevisiae*. In some embodiments, host cells are suitable mammalian cells. Many types of mammalian cells have been developed for expression vectors. Suitable mammalian cells include, but are not limited to, HEK293, Chinese hamster ovary cells (CHO), mouse myeloma cells, HeLa, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO-Rb50, HepG G2, DIKX-X11, J558L, young hamster kidney cells (BHK), and chicken embryo fibroblasts (CEF). Suitable host cells are further discussed in: Goeddel, *Gene Expression Technology: Methods in Enzymology*, 185, Academic Press, San Diego, California (1990). In some embodiments, suitable host cells are bovine cells, including but not limited to bovine embryonic stem cells, bovine induced pluripotent stem cells, bovine blastocyst cells, bovine spermatogonial stem cells, bovine oogonial cells, bovine primordial germ cells, bovine primordial germ cell-like cells, bovine totipotent cells, or other bovine cells described elsewhere herein.

[0359] In some embodiments, the vector may be a yeast expression vector. Examples of vectors used for expression in *Saccharomyces cerevisiae* include pYepSec1 (Baldari et al., 1987. *Journal of the European Society for Molecular Biology (EMBO J.)* 6:229-234), pMFa (Kuijan and Herskowitz, 1982. *Cell* 30:933-943), pJRY88 (Schultz et al., 1987. *Gene* 54:113-123), pYES2 (Invitrogen Corporation, San Diego, California) and picZ (Invitrogen Corporation, San Diego, California). As used herein, a “yeast expression vector” means a nucleic acid containing one or more sequences encoding RNA and / or polypeptides, and may further contain any desired elements controlling nucleic acid expression, and any elements enabling the expression vector to replicate and be maintained within yeast cells. Many suitable yeast expression vectors and their characteristics are known in the art; for example, various vectors and techniques are shown in the following literature: Yeast Protocols, 2nd edition, edited by Xiao, W. (Humana Press, New York, 2007) and Buckholz, RG and Gleeson, MA (1991) Biotechnology (New York) 9(11):1067-72. Yeast vectors may contain, but are not limited to, centromere (CEN) sequences, autonomous replication sequences (ARS), promoters such as RNA polymerase III promoters operatively linked to the sequence or gene of interest, terminators such as RNA polymerase III terminators, origin of replication, and marker genes (such as auxotrophs, antibiotics, or other optional markers). Examples of expression vectors for yeast may include plasmids, yeast artificial chromosomes, 2μ plasmids, yeast integration plasmids, yeast replication plasmids, shuttle vectors, and augmentative plasmids.

[0360] In some embodiments, the vector is a baculovirus vector or expression vector and can be adapted for expressing polynucleotides and / or proteins in insect cells. In some embodiments, suitable host cells are insect cells. Baculovirus vectors that can be used to express proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al., 1983. Molecular and Cell Biology 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39). rAAV (recombinant adeno-associated virus) vectors are preferably generated in insect cells, such as fall armyworm Sf9 insect cells grown in serum-free suspension cultures. Serum-free insect cells can be purchased from commercial suppliers, such as Sigma Aldrich (EX-CELL 405).

[0361] In some embodiments, the vector is a mammalian expression vector. In some embodiments, the mammalian expression vector is capable of expressing one or more polynucleotides and / or polypeptides in mammalian cells. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, 1987. *Nature* 329:840) and pMT2PC (Kaufman et al., 1987. *Journal of the European Society for Molecular Biology* 6:187-195). The mammalian expression vector may include one or more suitable regulatory elements capable of controlling the expression of one or more polynucleotides and / or proteins in mammalian cells. For example, commonly used promoters are derived from polyomaviruses, adenovirus 2, cytomegaloviruses, simian viruses 40, and other promoters disclosed herein and known in the art. Further details regarding suitable regulatory elements are provided elsewhere herein.

[0362] For other suitable expression vectors and vector systems for both prokaryotic and eukaryotic cells, see, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, Chapters 16 and 17.

[0363] In some embodiments, the vector may be a fusion vector or a fusion expression vector. In some embodiments, the fusion vector adds multiple amino acids to the protein encoded therein, such as to the N-terminus, C-terminus, or both of the recombinant protein. Such fusion vectors can be used for one or more purposes, such as: (i) increasing the expression of the recombinant protein; (ii) increasing the solubility of the recombinant protein; and (iii) aiding in the purification of the recombinant protein by acting as a ligand in affinity purification. In some embodiments, the expression of polynucleotides (such as non-coding polynucleotides) and proteins in prokaryotes can be performed in *E. coli*, wherein the vector contains a constitutive or inducible promoter that guides the expression of the fusion or non-fusion polynucleotide and / or protein. In some embodiments, the fusion expression vector may include a proteolytic cleavage site that may be introduced at the junction of the fusion vector backbone or other fusion portion with the recombinant polynucleotide or protein, such that the recombinant polynucleotide or protein can be isolated from the fusion vector backbone or other fusion portion after purification of the fusion polynucleotide or protein. Such enzymes and their homology recognition sequences include factor Xa, thrombin, and enterokinase. Examples of fusion expression vectors include pGEX (Pharmacia Biotech Inc.; Smith and Johnson, 1988. *Gene* 67:31-40), pMAL (New England Biolabs, Beverly, Massachusetts), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E protein, or protein A with the target recombinant protein, respectively. Examples of suitable inducible non-fusion *E. coli* expression vectors include pTrc (Amrann et al., (1988) *Gene* 69:301-315) and pET 11d (Studier et al., *Gene Expression Technology: Methods in Enzymatic Approaches*). ENZYMOLOGY, 185, Academic Press, San Diego, California (1990), 60-89.

[0364] In some embodiments, one or more vectors driving the expression of one or more elements of the genetic modification system described herein are introduced into a host cell such that the expression of the elements of the delivery system described herein is guided at one or more target sites at target polynucleotides to form a genetic modification system complex (e.g., a CRISPR-Cas complex), as in the target cell or the target cell genome. For example, the CRISPR-Cas effector proteins and nucleic acid components (e.g., guide polynucleotides) described herein may each be operatively linked to separate regulatory elements on separate vectors. RNA of different elements of a genetic modification (e.g., CRISPR-Cas) system can be delivered to animals, plants, microorganisms, or their cells to produce animals (e.g., mammals, such as cattle) constitutively, inducibly, or conditionally expressing different elements of a genetic modification (e.g., CRISPR-Cas) system described herein, said system incorporating one or more elements of a genetic modification system (e.g., a CRISPR-Cas system) described herein, or containing one or more cells incorporating and / or expressing one or more elements of a genetic modification (e.g., CRISPR-Cas) system described herein. Cell-free vectors and polynucleotide expression

[0365] In some embodiments, a polynucleotide encoding one or more features of a genetic modification system, or other polynucleotides described herein, can be expressed by a vector or suitable polynucleotide in a cell-free in vitro system. In other words, the polynucleotide can be transcribed in vitro and optionally translated. In vitro transcription / translation systems and suitable vectors are generally known in the art and are commercially available. Typically, in vitro transcription and in vitro translation systems replicate the processes of RNA and protein synthesis outside the cellular environment, respectively. Vectors and suitable polynucleotides used for in vitro transcription may include T7, SP6, T3, promoter regulatory sequences that can be recognized and acted upon by a suitable polymerase to transcribe the polynucleotide or vector.

[0366] In vitro translation can be standalone (e.g., translation of purified polynucleotides) or linked / coupled with transcription. In some embodiments, a cell-free (or in vitro) translation system may include extracts from rabbit reticulocytes, wheat germ, and / or *Escherichia coli*. The extracts may include various macromolecular components required for translating exogenous RNA (e.g., 70S or 80S ribosomes, tRNA, aminoacyl-tRNA, synthases, initiation factors, elongation factors, termination factors, etc.). Other components may be included or added during the translation reaction, including but not limited to amino acids, energy sources (ATP, GTP), energy regeneration systems (creatine phosphokinase and creatine phosphokinase (eukaryotic system)) (phosphoenolpyruvate and pyruvate kinase in bacterial systems), and other cofactors (Mg). 2+(e.g., K+, etc.). As previously mentioned, in vitro translation can be based on RNA or DNA starting materials. Some translation systems can utilize RNA templates as starting materials (e.g., reticulocyte lysates and wheat germ extracts). Some translation systems can utilize DNA templates as starting materials (e.g., E. coli-based systems). In these systems, transcription and translation are coupled, and DNA is first transcribed into RNA, which is then translated. Suitable standard and coupled cell-free translation systems are generally known in the art and are commercially available. Carrier characteristics

[0367] The vector may include additional features that can confer one or more functions upon the vector, the polynucleotide to be delivered, the virus or other particles generated therefrom (e.g., virus-like particles or exosomes), or the polypeptide expressed therein. Such features include, but are not limited to, regulatory elements, optional markers, molecular identifiers (e.g., molecular barcodes), stabilizing elements, etc. Those skilled in the art will understand that the design of the expression vector and the additional features included may depend on factors such as the selection of the host cell to be transformed, the desired expression level, etc. Adjustment element

[0368] In some embodiments, the polynucleotides and / or their vectors (such as the genetically modified system polynucleotides described herein) described herein may include one or more regulatory elements operatively linked to the polynucleotide. The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences), and cell localization signals (e.g., nuclear localization or output signals). Such regulatory elements are described, for example, in: Gene Expression Technology: Enzymatic Approaches, 185, Academic Press, San Diego, California (1990). Regulatory elements include elements that guide constitutive expression of a nucleotide sequence in many types of host cells and elements that guide expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may guide expression primarily in desired tissues of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements can also be directed in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, and this may or may not be tissue- or cell type-specific. In some embodiments, the vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5 or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5 or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5 or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, the U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rouss sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) (see, for example, Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the glycerol phosphokinase (PGK) promoter, and the EF1α promoter. The term “regulatory element” also encompasses enhancer elements such as WPRE; the CMV enhancer; the R-U5' region in the LTR of HTLV-I (Molecular and Cellular Biology, Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proceedings of the National Academy of Sciences, Vol. 78(3), pp. 1527-31, 1981).Exemplary promoters also include bovine U6 (bU6) and bovine 7SK (b7SK), as well as other bovine PolII promoters (see, for example, Lambeth et al., Animal Genetics, August 2006; 37(4):369-72), bovine papillomavirus-1 promoter (BPV-1) (Linz and Baker, Journal of Virology, August 1988; 62(8):2537-43. doi:10.1128 / JVI.62.8.2537-2543.1988), bovine SIX1 gene promoter (see, for example, Wei et al., Scientific Reports, Vol. 7, No. 12599(2017)), bovine growth hormone promoter (see, for example, Jiang et al., Nucleic Acids, Proteins, Enzymes and Molecular Genetics). (Gen.) 1999.274(12):7893-7900), bovine pyruvate carboxylase (see, for example, Hazelton et al., Journal of Dairy Science 91:91-99), bidirectional promoter (see, for example, Meersserman et al., DNA Research, Vol. 24, No. 3, June 2017, pp. 221-233), bovine Akt3 promoter (see, for example, Farmanullah et al., Journal of Genetic Engineering and Biotechnology (2021) 19:164), bovine α-lactalbumin promoter (see, for example, FEBS Letters) Lett.) 17 June 1991; 284(1):19-22), bovine β-casein promoter (see, for example, Cerdan et al., Molecular Propagation and Development, March 1998; 49(3):236-45), and any combination thereof.

[0369] In some embodiments, the regulatory sequence may be a regulatory sequence described in the following documents: U.S. Patent No. 7,776,321, U.S. Patent Publication No. 2011 / 0027239, or International Patent Publication No. WO 2011 / 028929, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the vector may contain a minimal promoter. In some embodiments, the minimal promoter is a Mecp2 promoter, a tRNA promoter, or a U6 promoter. In other embodiments, the minimal promoter is tissue-specific. In some embodiments, the length of the vector polynucleotide, the minimal promoter, and the polynucleotide sequence is less than 4.4 kb.

[0370] To express polynucleotides, vectors may include one or more transcription and / or translation initiation regulatory sequences, such as promoters, which direct the transcription of genes and / or the translation of encoded proteins in a cell. In some embodiments, constitutive promoters may be employed. Suitable constitutive promoters for mammalian cells are generally known in the art and include, but are not limited to, SV40, CAG, CMV, EF-1α, β-actin, RSV, and PGK. Suitable constitutive promoters for bacterial, yeast, and fungal cells, such as the T-7 promoter for bacterial expression and the alcohol dehydrogenase promoter for yeast expression, are known in the art.

[0371] In some embodiments, the regulatory ele...

Claims

1. A complemented non-human animal or embryo, the complemented non-human animal or embryo comprising: a first cell population comprising one or more cells, wherein the first cell population consists of engineered non-human animal cells or a population thereof comprising a NANOS3 genetic modification, wherein the NANOS3 genetic modification reduces or eliminates expression of a NANOS3 gene product; A second cell population comprising one or more cells, wherein the second cell population is not an engineered non-human cell or population thereof comprising a NANOS3 genetic modification, wherein the NANOS3 genetic modification reduces or eliminates expression of a NANOS3 gene product.

2. The complemented non-human animal or embryo of claim 1, wherein the second cell population comprises one or more engineered cells comprising one or more genetic modifications in one or more target genes, and wherein the one or more target genes are not NANOS3.

3. The complemented non-human animal or embryo of claim 1, wherein the second cell population does not comprise engineered cells.

4. The complemented non-human animal or embryo of claim 1, wherein the second cell population comprises an elite genome, a genomically selected genome, or both.

5. The complemented non-human animal or embryo of claim 1, wherein the second cell population comprises one or more a. embryonic cells, optionally fertilized eggs or inner cell mass cells; b. stem cells, optionally embryonic stem cells or induced pluripotent stem cells; c. Spermatogonial stem cells or oogonial stem cells; d. Primordial germ cells; or e. Primordial germ cell-like cells.

6. The complemented non-human animal or embryo of claim 1, wherein the second cell population is self-renewing cells.

7. The complemented non-human animal or embryo of claim 1, wherein the second cell population is pluripotent, totipotent or multipotent.

8. The complemented non-human animal or embryo of claim 1, wherein the second cell population is germline competent.

9. The complemented non-human animal or embryo of claim 1, wherein the complemented embryo is a preimplantation embryo, optionally a fertilized egg, a 2-cell, a 4-cell, an 8-cell, a 16-cell, a blastocyst or a morula.

10. The complemented non-human animal or embryo of claim 1, wherein the first cell population comprises a percentage of the cells of the complemented non-human animal or embryo ranging from about 25% to any percentage up to but not including 100%.

11. The complemented non-human animal or embryo of claim 1, wherein the complemented non-human animal or embryo comprises at least one cell from the second cell population, optionally wherein the second cell population comprises a percentage of the cells of the engineered non-human animal or embryo ranging from any non-zero percentage to about 75%.

12. The complemented non-human animal or embryo of claim 1, wherein the complemented embryo is a day 3 post fertilization (PF) embryo, a day 4 post fertilization (PF) embryo, a day 5 post fertilization (PF) embryo, or a day 6 post fertilization (PF) embryo.

13. The complemented non-human animal or embryo according to claim 12, wherein a. The complemented embryo on day 3 after fertilization comprises about 5 cells from the second cell population; b. The complemented embryo on day 4 after fertilization comprises about 5 cells from the second cell population; c. The complemented embryo at day 5 after fertilization comprises about 8-10 cells from the second cell population; and / or d. The complemented embryo at day 6 after fertilization contains about 10-20 cells from the second cell population.

14. The complemented non-human animal or embryo of claim 1, wherein the complemented embryo is a morula.

15. The complemented non-human animal or embryo of claim 1, wherein the complemented non-human animal or embryo is male.

16. The complemented non-human animal embryo of claim 1, wherein the complemented non-human animal or embryo is female.

17. The complemented non-human animal or embryo of claim 1, wherein (a) the complemented non-human animal or embryo is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, mouse, hare or guinea pig; (b) the engineered non-human animal cell or colony thereof is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, mouse, hare or guinea pig cell; (c) the first cell colony comprising one or more cells is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, mouse, hare or guinea pig cell colony; (d) the second cell colony comprising one or more cells is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, mouse, hare or guinea pig cell colony; or (e) any combination of (a)-(d).

18. The complemented non-human animal or embryo of claim 1, wherein the NANOS3 gene modification is a. Insertion of one or more nucleotides; b. Deletion of one or more nucleotides; c. substitution of one or more nucleotides; or d. Any combination of (a)-(c).

19. The complemented non-human animal or embryo of claim 1, wherein the NANOS3 gene modification is located in exon 1 of the NANOS3 gene, optionally in the zinc finger domain of the NANOS3 gene.

20. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cells or population thereof are monoallelic or biallelic with respect to the NANOS3 gene modification.

21. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cells or populations thereof do not express a functional NANOS3 gene or gene product.

22. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cells are heterozygous or homozygous for the NANOS3 genetic modification, wherein the NANOS3 genetic modification is optionally a NANOS3 gene knockout.

23. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cell or population thereof is an engineered male cell or population thereof.

24. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cell is an engineered female cell or cell population.

25. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cell or population thereof is an engineered somatic cell or population thereof.

26. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cell or population thereof is an engineered germ cell or population thereof.

27. The complemented non-human animal or embryo of claim 26, wherein the engineered germ cell or population thereof is an engineered gamete or population thereof.

28. The complemented non-human animal or embryo of claim 27, wherein the engineered gametes or populations thereof are engineered sperm or populations thereof or engineered eggs or populations thereof.

29. The complemented non-human animal or embryo of claim 26, wherein the engineered germ cell or population thereof is an engineered immature germ cell or population thereof.

30. The complemented non-human animal or embryo of claim 29, wherein the engineered immature germ cells or populations thereof are engineered sperm cells or populations thereof or engineered oocytes or populations thereof.

31. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cells are a population of engineered embryonic cells thereof, optionally wherein the engineered embryonic cells are fertilized eggs.

32. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cell population thereof is an engineered blastocyst cell or population thereof, optionally an engineered inner cell mass cell or population thereof.

33. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cell or population thereof is an engineered stem cell or population thereof, optionally an engineered embryonic stem cell or population thereof or an induced pluripotent stem cell or population thereof.

34. The complemented non-human animal or embryo of claim 26, wherein the engineered non-human animal cell or cell population is an engineered spermatogonial stem cell or population thereof or an engineered oogonial stem cell or population thereof.

35. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cell or cell population cell is a primordial germ cell or population thereof or an engineered primordial germ cell-like cell or population thereof.

36. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cell or population thereof is an engineered self-renewing cell or population thereof.

37. The complemented non-human animal or embryo of claim 1, wherein the engineered non-human animal cells are pluripotent, totipotent or multipotent.

38. A non-human animal developed or produced from the complemented non-human animal or embryo of claim 1.

39. The non-human animal of claim 38, wherein one or more germ cells of the engineered animal are derived from the second cell population.

40. The non-human animal of claim 38, wherein between about 0.001% and 100% of said germ cells are derived from said second cell population.

41. The non-human animal of claim 38, wherein the non-human animal is male or female.

42. A non-human animal or progeny of one or more complemented non-human animals according to claim 1.

43. An engineered non-human animal cell or population thereof, the engineered non-human animal cell or population thereof comprising: A NANOS3 gene modification, wherein the NANOS3 gene modification reduces or eliminates expression of a NANOS3 gene product.

44. The engineered non-human animal cell or population thereof of claim 43, wherein the NANOS3 gene modification is a. Insertion of one or more nucleotides; b. Deletion of one or more nucleotides; c. Substitution of one or more nucleotides; d. Or any combination of (a)-(c).

45. The engineered non-human animal cell or population thereof of claim 43, wherein the NANOS3 gene modification is located in exon 1 of the NANOS3 gene, optionally in the zinc finger domain of the NANOS3 gene.

46. ​​The engineered non-human animal cell or population thereof of claim 43, wherein the engineered non-human animal cell or population thereof is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, mouse, rabbit or guinea pig cell.

47. The engineered non-human animal cell or population thereof of claim 43, wherein one or both of the NANOS3 alleles are modified.

48. The engineered non-human animal cell or population thereof of claim 43, wherein the engineered non-human animal cell or population thereof is monoallelic or biallelic with respect to the NANOS3 gene modification.

49. The engineered non-human animal cell or population thereof according to claim 43, wherein the engineered non-human animal cell population thereof does not express a functional NANOS3 gene or gene product.

50. The engineered non-human animal cell or population thereof of claim 43, wherein the engineered non-human animal cell is heterozygous or homozygous for the NANOS3 gene modification, wherein the NANOS3 gene modification is optionally a NANOS3 gene knockout.

51. The engineered non-human animal cell or population thereof of claim 43, wherein the engineered non-human animal cell or population thereof is an engineered male cell or population thereof.

52. The engineered non-human animal cell or population thereof of claim 43, wherein the engineered non-human animal cell is an engineered female cell or cell population.

53. The engineered non-human animal cell or population thereof of claim 43, wherein the engineered non-human animal cell or population thereof is an engineered somatic cell or population thereof.

54. The engineered non-human animal cell or population thereof of claim 43, wherein the engineered non-human animal cell or population thereof is an engineered germ cell or population thereof.

55. The engineered non-human animal cell or cell population of claim 54, wherein the engineered germ cell or population thereof is an engineered gamete or population thereof.

56. An engineered non-human animal cell or cell population according to claim 55, wherein the engineered gamete or population thereof is an engineered sperm or population thereof or an engineered egg or population thereof.

57. The engineered non-human animal cell or population thereof of claim 54, wherein the engineered germ cell or population thereof is an engineered immature germ cell or population thereof.

58. The engineered non-human animal cell or population thereof of claim 57, wherein the engineered immature germ cells or population thereof are engineered sperm cells or populations thereof or engineered oocytes or populations thereof.

59. The engineered non-human animal cell or population thereof of claim 43, wherein the engineered non-human animal cell is a population of engineered embryonic cells thereof, optionally wherein the engineered embryonic cells are fertilized eggs.

60. The engineered non-human animal cell or population thereof of claim 43, wherein the engineered non-human animal cell population thereof is an engineered blastocyst cell or population thereof, optionally an engineered inner cell mass cell or population thereof.

61. The engineered non-human animal cell or population thereof of claim 43, wherein the engineered non-human animal cell or population thereof is an engineered stem cell or population thereof, optionally an engineered embryonic stem cell or population thereof or an induced pluripotent stem cell or population thereof.

62. The engineered non-human animal cell or cell population of claim 43, wherein the engineered non-human animal cell or cell population is an engineered spermatogonial stem cell or a population thereof or an engineered oogonial stem cell or a population thereof.

63. The engineered non-human animal cell or population thereof of claim 43, wherein the engineered non-human animal cell or cell population cells are primordial germ cells or populations thereof or engineered primordial germ cell-like cells or populations thereof.

64. The engineered non-human animal cell or population thereof of claim 43, wherein the engineered non-human animal cell or population thereof is an engineered self-renewing cell or population thereof.

65. The engineered non-human cell of claim 43, wherein the engineered non-human animal cell is pluripotent, totipotent or multipotent.

66. An engineered non-human animal, embryo, or progeny thereof, comprising an engineered non-human animal cell or a population thereof according to claim 43.

67. The engineered non-human animal, embryo, or progeny thereof of claim 66, wherein the engineered non-human animal, embryo, or progeny thereof is a chimera.

68. The engineered non-human animal, embryo, or progeny thereof of claim 66, wherein the engineered non-human animal, embryo, or progeny thereof is a mosaic.

69. The engineered non-human animal, embryo, or progeny thereof of claim 66, wherein the engineered non-human animal, embryo, or progeny thereof is not chimeric.

70. The engineered non-human animal, embryo, or progeny thereof of claim 66, which is not a mosaic.

71. The engineered non-human animal, embryo, or progeny thereof of claim 66, wherein at least 1 cell or at least 0.0001% to 100% of all cells of the engineered non-human animal, embryo, or progeny thereof are engineered non-human animal cells comprising a NANOS3 gene modification, wherein the NANOS3 gene modification reduces or eliminates expression of a NANOS3 gene product.

72. The engineered non-human animal, embryo, or progeny thereof of claim 66, wherein the engineered non-human animal, embryo, or progeny thereof is male.

73. The engineered non-human animal, embryo, or progeny thereof of claim 66, wherein the engineered non-human animal, embryo, or progeny thereof is female.

74. The engineered non-human animal, embryo, or progeny thereof of claim 66, wherein the engineered non-human animal is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, mouse, rabbit, or guinea pig.

75. The engineered non-human animal, embryo, or progeny thereof of claim 66, further comprising a second cell population comprising one or more cells, wherein the second cell population does not comprise engineered non-human animal cells comprising a NANOS3 gene modification, wherein the NANOS3 gene modification reduces or eliminates expression of the NANOS3 gene product, and wherein the second cell population is germline competent cells, germ cells, or gametes.

76. The engineered non-human animal, embryo, or progeny thereof of claim 75, wherein the second cell population comprises or consists of: One or more a. embryonic cells, optionally fertilized eggs or inner cell mass cells; b. stem cells, optionally embryonic stem cells or induced pluripotent stem cells; c. Spermatogonial stem cells or oogonial stem cells; d. Primordial germ cells; or e. Primordial germ cell-like cells.

77. The engineered non-human animal, embryo, or progeny thereof of claim 75, wherein the second cell population comprises or consists of one or more sperm cells or one or more oocytes.

78. The engineered non-human animal, embryo, or progeny thereof of claim 75, wherein the second cell population comprises or consists of sperm or eggs.

79. The engineered non-human animal, embryo, or progeny thereof of claim 75, wherein the second cell population comprises or consists of one or more engineered cells comprising one or more genetic modifications in one or more target genes, and wherein the one or more target genes are not NANOS3.

80. The engineered non-human animal, embryo, or progeny thereof of claim 75, wherein the second cell population does not comprise or consist of engineered cells or a population thereof.

81. An engineered non-human animal, embryo, or progeny thereof according to claim 80, wherein the second cell population comprises or consists of an elite genome, a genomically selected genome, or both.

82. A method of producing a non-human animal or embryo modified with NANOS3, the method comprising: introducing one or more NANOS3 genetic modifications into a non-human animal cell, wherein the NANOS3 genetic modification reduces or eliminates expression of a NANOS3 gene product; and One or more of the following techniques: somatic cell nuclear transfer, oocyte pronuclear DNA microinjection, zygote microinjection or embryo microinjection, intracytoplasmic sperm injection, in vitro fertilization, embryo transfer, in vitro embryo culture, or any combination thereof.

83. The method of claim 82, wherein the NANOS3 gene modification is a. Insertion of one or more nucleotides; b. Deletion of one or more nucleotides; c. substitution of one or more nucleotides; or d. Any combination of (a)-(c).

84. The method of claim 82, wherein the NANOS3 gene modification is located in exon 1 of the NANOS3 gene, optionally in the zinc finger domain of the NANOS3 gene.

85. The method of claim 82, wherein one or both of the NANOS3 alleles are modified.

86. The method of claim 82, wherein the non-human animal or embryo is monoallelic or biallelic with respect to the NANOS3 genetic modification.

87. The method of claim 82, wherein the engineered non-human animal or embryo does not express a functional NANOS3 gene or gene product.

88. The method of claim 82, wherein the non-human animal or embryo is heterozygous or homozygous for a NANOS3 knockout.

89. The method of claim 82, wherein the non-human animal or embryo is germline ablated.

90. The method of claim 82, wherein the non-human animal or embryo is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, mouse, rabbit or guinea pig.

91. The method of claim 82, wherein the non-human animal or embryo is male.

92. The method of claim 82, wherein the non-human animal or embryo is female.

93. The method of claim 82, wherein introducing one or more NANOS3 gene modifications into the non-human animal cells comprises CRISPR-Cas-mediated gene modification, zinc finger nuclease gene modification, TALEN-mediated gene modification, recombinase-mediated gene modification, lead editing-mediated gene modification, meganuclease-mediated gene modification, transposase / transposon-mediated gene modification, or any combination thereof.

94. The method of claim 93, wherein introducing one or more NANOS3 gene modifications into the non-human animal cells comprises using a CRISPR-Cas system, and wherein the guide RNA of the CRISPR-Cas system targets exon 1 of the NANOS3 gene, optionally in the zinc finger region, and is optionally selected from any one of SEQ ID NOs: 39-45 or any combination thereof.

95. A method for non-human animal embryo complementation, the method comprising: Optionally, introducing the self-renewing exogenous cell population into a non-human animal preimplantation embryo on about day 3, day 4, day 5, or day 6 after fertilization; optionally washing the non-human animal preimplantation embryos in HEPES or other suitable buffer; and The non-human preimplantation embryos are cultured in a suitable culture medium, optionally consisting of a suitable bovine culture medium supplemented with at least N2, B27, FGF and IWR-1 in a 1:1 volume ratio.

96. The method of claim 95, wherein the number of exogenous cells introduced is from about 1 to about 25 cells, or about 30-50% of the total number of cells present in the embryo prior to the introduction of the exogenous cells.

97. The method of claim 95, wherein a. The number of exogenous cells introduced 3 or 4 days after fertilization is about 5 cells; b. wherein the number of exogenous cells introduced at 5 days after fertilization is about 8, 9 or 10 cells; or c. The number of exogenous cells introduced at 6 days after fertilization was approximately 10-20 cells.

98. The method of non-human animal embryo complementation according to claim 95, wherein the self-renewing exogenous cells are embryonic stem cells, expanded embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, multipotent stem cells, totipotent stem cells, primordial germ cells, primordial germ cell-like cells, totipotent cells or a combination thereof.

99. A method for non-human animal embryo complementation according to claim 95, wherein the non-human animal embryo is genetically germline ablated.

100. The method of non-human animal embryo complementation according to claim 95, wherein the non-human animal embryo comprises or consists of one or more engineered non-human animal cells, and the one or more engineered non-human animal cells comprise a NANOS3 gene modification, wherein the NANOS3 gene modification reduces or eliminates the expression of the NANOS3 gene product.

101. The method of non-human animal embryo complementation of claim 95, wherein the self-renewing exogenous cells are germline competent.

102. The method of non-human animal embryo complementation of claim 95, wherein the self-renewing exogenous cells are engineered cells comprising one or more genetic modifications in one or more target genes, and wherein the one or more target genes are not NANOS3.

103. The method of non-human animal embryo complementation of claim 95, wherein the self-renewing exogenous cells are not genetically modified.

104. The method of non-human animal embryo complementation of claim 95, wherein the self-renewing exogenous cells comprise an elite genome, a genomically selected genome, or both.

105. The method of non-human animal embryo complementation according to claim 95, wherein the non-human animal embryo is a cow, horse, pig, sheep, goat, camel, deer, dog, cat, mouse, rabbit or guinea pig.

106. A complemented non-human embryo produced by the method of embryo complementation according to claim 95.

107. A non-human animal produced by the embryo according to claim 107 and its offspring.

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