Methods and compositions for targeted genetic inactivation of primordial germ cell (PGC) specification genes for efficient germline ablation and exclusive donor cell germline transmission in gene-edited animals

Abstract

The present application provides Methods and Compositions for Targeted Genetic Inactivation of NANOS for efficient germline ablation and exclusive donor cell germline transmission in non-human mammals. The application of this technique in the agricultural industry holds significant promise for accelerating the propagation of desirable traits in agricultural animals and catalyzing the efficient use of valuable genetic resources and the creation of genetically uniform herds, thereby improving the overall sustainability and profitability of livestock farming.

Description

Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)METHODS AND COMPOSITIONS FOR TARGETED GENETIC INACTIVATION OF PRIMORDIAL GERM CELL (PGC) SPECIFICATION GENES FOR EFFICIENT GERMLINE ABLATION AND EXCLUSIVE DONOR CELL GERMLINE TRANSMISSION IN GENE-EDITED ANIMALSCROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63,744,754, filed January 13, 2025, the content of which is incorporated herein by reference in its entirety for any and all purposes.TECHNICAL FIELD[00021 The technology relates to the field of gene editing.BACKGROUND

[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] The development of genetically engineered animals often necessitates precise germline modifications to ensure the desired traits are inherited by subsequent generations. However, introducing genetically modified pluripotent cells — whether derived from embryos, embryonic stem (ES) cells, induced pluripotent stem cells (iPS), or embryonic germ cells (EGC) — frequently leads to challenges in achieving germline transmission. Specifically, these modified cells may fail to colonize the germline effectively, resulting in gene-edited offspring that do not transmit the desired genetic modifications.

[0005] Traditional methods have outlined creating sterile recipient animals to support germline transplantation by focusing on spermatogonial stem cell (SSC) transfer techniques. These methods are associated with several critical limitations that reduce their effectiveness and scalability.

[0006] Firstly, these methods emphasize the use of chemotoxic drugs or irradiation to eliminate endogenous germline cells in recipient animals. These treatments, although partially effective, introduce significant challenges. Chemotoxic agents often result in14899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)systemic toxicity, affecting not only germline cells but also bone marrow and other stem cell populations, leading to unintended consequences for animal health.

[0007] Secondly, the disposal of biohazardous waste, such as feces and urine containing residual chemotoxic agents, creates logistical and environmental hurdles.Irradiation, while avoiding some of the systemic toxicity of chemotoxic agents, can still damage testicular support cells, such as Sertoli and Leydig cells, thereby compromising the recipient’s ability to support donor-derived spermatogenesis.

[0008] Thirdly, these prior methods fail to address the persistent presence of endogenous germ cells in recipient animals, even after treatments. This residual population of endogenous germ cells competes with donor cells, often leading to incomplete donor cell engraftment and the generation of mixed germline chimerism. Consequently, the efficiency of germline transmission from donor cells is significantly reduced, and the process of creating animals with exclusively donor-derived germlines becomes highly unreliable.

[0009] Lastly, these methods rely heavily on the use of SSCs as donor cells, which introduces additional challenges. Isolating, expanding, and transplanting SSCs in large domestic animals like porcine or bovine is technically complex, resource-intensive, and prone to variability. These challenges limit the scalability and broad applicability of the approach, particularly in agricultural settings where efficiency and reproducibility are critical.

[0010] In contrast, the present application addresses these limitations by fundamentally shifting the paradigm of germline ablation and donor cell colonization. For example, the SSC techniques focus on preparing recipient animals via chemotoxic or irradiation-based methods, whereas this application utilizes gene editing and embryo complementation to ensure that no residual endogenous germ cells persist in the host embryos. This creates an optimized environment where donor cells can engraft exclusively, resulting in germline chimeras with 100% donor-derived germlines.

[0011] Additionally, the use of genetically inactivated host embryos simplifies the overall process, making it more scalable and efficient for use in agricultural biotechnology.24899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0012] Furthermore, the ability to complement embryos through blastomere or inner cell mass (ICM) transplantation provides opportunities to create gene-edited animals with desired genetic profiles as opposed to creating sterile recipient animals which require postnatal transplantation.

[0013] Finally, unlike the SSC techniques, this application enables the ability to utilize donor cells from any desired genotype, derived by various methods (for example, embryonic stem cell nuclear transfer (ESCNT) complementation, IVF, embryo flushing) in a male host embryo. This is a significant advancement that addresses several critical challenges, including single-sex sperm production, dam line genetic dissemination, and trait dissemination, for example in producing gene-edited animals. This flexibility allows researchers to overcome sex-specific limitations in donor cell availability and compatibility, which is particularly important when the desired genetic traits or modifications are present in donor cells of a specific sex.

[0014] Additionally, using female donor cells in a male host embryo provides unique opportunities to study and manipulate germline development in cross-sex environments, offering insights into sex-biased epigenetic reprogramming and germline restoration. This capability expands the range of potential applications, including the generation of male gene-edited animals carrying specific maternal traits or genetic edits. By enabling cross-sex complementation, this application enhances efficiency and versatility in breeding programs, conservation efforts, and biotechnological applications, paving the way for more precise and customizable animal production strategies.

[0015] Also, NANOS genes, such as NANOS2 and NANOS3, are known to be the most highly-conserved across species and are role-specific to PGC maintenance and development.

[0016] These characteristics allow PGC specification genes, such as NANOS, to enhance utility as targets in a wide range of non-human animals, while also achieving robust germline modifications with greater predictability and fewer unintended consequences compared to targeting other PGC specification genes.34899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)SUMMARY

[0017] In one aspect, the present disclosure provides a method for producing a nonhuman gene-edited embryo with donor-derived germ cells, the method comprising: providing a host embryo comprising an inactivated PGC specification gene;and complementing the host embryo with donor cells to yield the gene-edited embryo, wherein the germ cells of the gene-edited embryo are exclusively derived from the donor. In some embodiments, the inactivated PGC specification gene is NANOS. In some embodiments, the inactivated PGC specification gene is NANOS2. In some embodiments, the host embryo is complemented at any stage from fertilized zygote to blastocyst stage. In some embodiments, the host embryo is complemented at the morula stage as an early-stage embryo. In some embodiments, the donor cells comprise one or more pluripotent cells. In some embodiments, the one or more pluripotent cells comprise embryonic stem cells or induced pluripotent stem cells. In some embodiments, the non-human animal is a livestock animal. In some embodiments, the non-human animal is a bovine.|0018] In some embodiments, the inactivated PGC specification gene is NANOS2, and wherein the NANOS2 is inactivated by introducing into the cell an engineered CRISPR / Cas system comprising a Cas protein or a polynucleotide encoding the Cas protein, and at least one guide RNA (gRNA) or a polynucleotide encoding the gRNA, wherein the gRNA specifically hybridizes to a NANOS2 sequence. In some embodiments, the at least one gRNA is selected from the group consisting SEQ ID NO: 1-12. In some embodiments, the at least one gRNA comprises or consists of a gRNA pair. In some embodiments, the gRNA pair is selected from the group consisting of: (a) gRNA 370 (SEQ ID NO: 11) and gRNA 371 (SEQ ID NO: 12); (b) gRNA 360 (SEQ ID NO: 1) and gRNA 367 (SEQ ID NO: 8); (c) gRNA 361 (SEQ ID NO: 2) and gRNA 367 (SEQ ID NO: 8); and (d) gRNA 363 (SEQ ID NO: 4) and gRNA 368 (SEQ ID NO: 9). In some embodiments, the Cas protein is selected from spCas9 and OpenCRISPR-1 (OC-1). In some embodiments, the Cas protein is SpCas9. In some embodiments, the Cas protein is OC-1.

[0019] In some embodiments, the donor cells are XX-chromosome-bearing cells.44899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0020] In some embodiments, the method further comprises creating a non-human gene-edited animal by implanting the gene-edited embryo into a surrogate mother and allowing the embryo to develop to term. In some embodiments, the non-human gene-edited animal is viable and fertile.

[0021] In one aspect, the present disclosure provides a non-human gene-edited embryo comprising host cells and donor cells, wherein the host cells comprise an inactivated primordial germ cell (PGC) specification gene, and wherein the donor cells exclusively contribute to the germ cells of the non-human gene-edited embryo.

[0022] In one aspect, the present disclosure provides a non-human gene-edited embryo comprising host cells and donor cells, wherein the host cells comprise an inactivated NANOS gene, and wherein the donor cells exclusively contribute to the germ cells of the non-human gene-edited embryo.

[0023] In one aspect, the present disclosure provides a non-human gene-edited embryo comprising host cells and donor cells, wherein the host cells comprise an inactivated NANOS2 gene, and wherein the donor cells exclusively contribute to the germ cells of the non-human gene-edited embryo.

[0024] In one aspect, the present disclosure provides a non-human gene-edited animal produced by the method of any one of the foregoing embodiments, wherein the non-human gene-edited animal possesses a germline exclusively derived from the donor cells.

[0025] In one aspect, the present disclosure provides a biological specimen obtained from the non-human gene-edited animal of any of the foregoing embodiments.

[0026] In one aspect, the present disclosure provides a method for producing a gene-edited bovine embryo with donor-derived bovine germ cells, the method comprising complementing a host embryo comprising an inactivated NANOS2 gene with XX-chromosome-bearing donor cells to yield the gene-edited bovine embryo, wherein the germ cells of the gene-edited embryo are exclusively derived from the donor.54899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0027] In one aspect, the present disclosure provides a method for genotyping the non-human gene-edited embryo of any of the foregoing embodiments, the method comprising: obtaining a biological sample from the embryo; extracting nucleic acids from the sample; and performing polymerase chain reaction (PCR) analysis to confirm the presence of the inactivated PGC specification gene and donor-derived genetic markers. In some embodiments, the PCR analysis includes amplification of sequences flanking the inactivated PGC specification gene to verify the gene-editing event. In some embodiments, the PCR analysis includes real-time PCR to quantify the relative abundance of donor-derived genetic material.

[0028] In one aspect, the present disclosure provides a method for genotyping the non-human gene-edited animal of any of the foregoing embodiments, the method comprising: obtaining a biological specimen from the animal; extracting nucleic acids from the specimen; and performing PCR analysis to confirm the donor-derived germline contribution. In some embodiments, the PCR analysis includes amplification of sequences flanking the inactivated PGC specification gene to verify the gene-editing event.

[0029] In one aspect, the present disclosure provides a method for producing a gene-edited bovine embryo with donor-derived bovine germ cells, the method comprising complementing a host embryo comprising an inactivated NANOS2 gene with XX-chromosome-bearing donor cells to yield the gene-edited bovine embryo, wherein the germ cells of the gene-edited embryo are exclusively derived from the donor.

[0030] In one aspect, the present disclosure provides a gene-edited bovine embryo produced by the method of any of the foregoing embodiments.

[0031] In one aspect, the present disclosure provides a bovine animal produced by the method of any of the foregoing embodiments.

[0032] In one aspect, the present disclosure provides a biological specimen obtained from the bovine animal of any of the foregoing embodiments.

[0033] Both the foregoing summary and the following description of the drawings and detailed description are exemplary and explanatory. They are intended to provide further64899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)details of the disclosure, but are not to be construed as limiting. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the disclosure.

[0034] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below are provided as being part of the inventive subject matter disclosed herein and may be employed in any combination to achieve the benefits described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 illustrates the process of embryo complementation, showcasing two distinct experimental setups. The upper section details the integration of blastomeres from a Day 5 donor embryo into host male embryos (NANOS2 ) at the same developmental stage. The lower section describes isolating the inner cell mass (ICM) from a Day 7 donor embryo and transferring it into a Day 7 host male embryo (NANOS2 ). Both approaches culminate in embryo transfer to a recipient cow, aimed at successful embryo development.

[0036] FIG. 2 illustrates the results of Inner Cell Mass (ICM) complementation using male embryonic stem cell nuclear transfer (ESCNT) into a NANOS2 male host embryo. The left side depicts an early-stage embryo on Day 8, showcasing the green fluorescence from GFP-labeled male ICM cells that have successfully integrated into the host environment. The right side presents a more developed embryo on Day 14, exhibiting distinct structures such as the Embryonic Disc and Trophectoderm, all highlighted in green (displayed as light grey) due to the expression of GFP from transplanted cells.

[0037] FIG. 3 illustrates two fluorescent microscopy images of D90 fetal testes. The left image displays green cells (displayed as white) from male ESCNT ICM incorporated into a NANOS2 male host embryo, with a positive recovery rate of 4 out of 4 fetuses. The right image shows red cells (displayed as white) from female ESCNT blastomeres in the same type of host embryo, with a recovery rate of 1 out of 4 fetuses.

[0038] FIG. 4 displays a fluorescent microscopy image of D90 fetal testes showcasing GFP (green fluorescent protein) incorporation. The image reveals highlighted74899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)green (displayed as light grey) donor cells within a cross-section of the seminiferous tubules, with white arrows indicating specific areas of interest.

[0039] FIG. 5 depicts a fluorescent microscopy image of D90 fetal testes, highlighting RFP (red fluorescent protein) expression in female donor cells. The image shows a cross-section of seminiferous tubules, with white arrows indicating specific RFP-expressing cells. Results indicate successful incorporation of these cells in the developing testicular tissue.

[0040] FIG. 6 presents an exemplary wild-type (WT) target sequence of the bovine NANOS2 gene, designated as NANOS2_NM_001281904.1_Target sequence (SEQ ID NO: 37). The sequence is essential for researchers investigating the genetic traits and functions of this gene in bovine species. It is displayed as a continuous string of nucleotide bases, consisting of adenine (A), cytosine (C), guanine (G), and thymine (T), which collectively form the genetic code for this segment of the genome. The representation showcases the intricate arrangement of the nucleotides, which is crucial for the design of guide RNAs in applications such as CRISPR gene editing. The underlined sequence represents an exemplary target sequence and protospacer adjacent motif (PAM) that can be targeted by gRNA 370 (SEQ ID NO: 11). The bolded sequence represents an exemplary target sequence and protospacer adjacent motif (PAM) that can be targeted by gRNA 371 (SEQ ID NO: 12).

[0041] FIG. 7 represents an exemplary gene-edited sequence which results from targeting the wild type NANOS2 sequence from FIG. 6 with the dual guide pair 370-371.

[0042] FIG. 8 shows a schematic and a panel of images illustrating the successful generation of chimeric bulls by embryo complementation. In the schematic, lighter gray cells represent XX-chromosome-bearing donor cells. Such cells are also surrounded by a dotted black line. The images in the panel are fluorescent microscopy images of postnatal testes at 5 months from two chimeric bulls, Calf 30 and Calf 32, showcasing RFP (red fluorescent protein, as indicated by the bright white cells encircled with a dotted white line) incorporation.84899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)DETAILED DESCRIPTION

[0043] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology. The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present technology belongs.I. Definitions

[0044] As used herein, “induced pluripotent cells” means embryonic-like cells that are reprogrammed from adult cells to an immature phenotype. There are several methods known in the art, such as “a simple new way to independent: acid.” published in Nature 1 / 29 2014, science date.com / releases / 2014 / 01 / 140129184445 last visited 2 / 52014, and U.S.Patent Publication No. 2010 / 0041054. It should be understood that other pluripotent cells including blastocyst like structures, such as blastoids, as well as parthenogenetic embryo, can be used in replacement or in addition to other pluripotent cell types.

[0045] The term “parthenogenetic stem cells” refers to parthenogenetically activated stem cells derived from eggs. Methods of generating parthenogenetic stem cells are known in the art. See, e.g., Cibelli et al. (2002) Science 295(5556):819 and Vrana et al. (2003) Proc. Natl. Acad. Sci. USA 100(suppl. 1) 11911-6.

[0046] The term “stem cell” refers to a cell that is in an undifferentiated or partially differentiated state and has the ability to self-renew and / or produce differentiated progeny. “Self-renewal” is defined as the ability of a stem cell to proliferate and produce more of such stem cells while maintaining its developmental potential (i.e., totipotent, pluripotent, multifunctional, etc I). Stem cells also include, but is not limited to, spermatogonial stem cells, which have the ability to self-renew and differentiate into gametes. Spermatogonial stem cells can be created and utilized in various applications, as outlined in US Patent No. 10,280,397 B2.94899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0047] The term “somatic stem cell” as used herein refers to any stem cell derived from non-embryonic tissues, including fetal, juvenile and adult tissues. Natural somatic stem cells have been isolated from a wide variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Exemplary naturally occurring somatic stem cells include, but are not limited to, Mesenchymal Stem Cells (MSCs) and Neural Stem Cells (NSCs). In some embodiments, the stem or progenitor cells can be embryonic stem cells.

[0048] As used herein, “embryo” encompasses all stages from a zygote through early preimplantation stages, including cleavage, morula, and blastocyst stages, regardless of the method of generation (e.g., natural fertilization, in vitro fertilization, parthenogenesis, somatic cell nuclear transfer, or other reproductive technologies). The term “early-stage embryo” means any embryo at embryonic stages between fertilized ovum and blastocyst. As used herein, “embryonic stem cells” refer to stem cells derived from tissue that is postfertilized but formed prior to the end of pregnancy, including pre-embryonic tissue (e.g., blastocyst), embryonic tissue, or fetal tissue, taken at any time during pregnancy. Most commonly, embryonic stem cells are pluripotent cells derived from early embryos or blastocysts. Embryonic stem cells can be obtained directly from suitable tissues, including but not limited to human tissues, or from established embryonic cell lines, “embryonic stemlike cell” refers to a cell that has one or more, but not all, of the characteristics of an embryonic stem cell.

[0049] As used herein, the term “biological specimen” refers to any material originating from, derived from, or obtained from a living or previously living organism, including but not limited to whole organisms, body parts, cells, tissues, organs, and biological fluids. A biological specimen can include reproductive materials such as sperm, seminal fluid, ejaculate, ova, oocytes, embryos, and other gametes, as well as any fractions, isolates, derivatives, lysates, or preparations thereof.

[0050] As used herein, “animal cell” means a cell, including, but not limited to, a somatic cell, culture cell, gamete cell, blood cell, zygote, and embryonic cell. These animal cells can be reproductive or non-reproductive cells. As used herein, cells may be isolated from an animal or embryo and maintained in tissue culture. Also included are mixed cultures104899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)that can comprise gene-edited cells of the present specification and a non-gene-edited support cell or feeder cell.

[0051] As used herein, the term “livestock animal” includes any animal traditionally raised in livestock farming, for example artiodactyls such as bovine (e.g., cattle) or porcine (e.g., pigs) animals, avian animals such as chickens, turkeys, ducks, geese, guinea fowl, or squabs, or equine animals such as horses or donkeys. The term “livestock animal” does not include rats, mice, or other rodents.

[0052] As used herein, the term “non-human animal” refers to all animals excluding species of the genus Homo.

[0053] As used herein, “mating” or “breeding” refers to the process by which genetic material from two parental sources is combined to produce offspring, encompassing all natural and artificial methods that facilitate the union of gametes (e.g., sperm and egg) or their equivalent. As used herein, the preferred meaning of “fertilization” as used herein to refer to non-human animal breeding encompasses any technique that produces a viable embryo. In vitro fertilization (IVF) can be the harvesting of unfertilized oocytes(s) and the subsequent fertilization of those oocytes with semen in vitro (ie., in the laboratory) instead of in vivo (z.e., in the live animal ) as in standard ET. The fertilized oocytes(s) or embryo(s) from the oocyte donor can then be transferred into another female (embryo recipient).Embryo transfer (ET) can be the harvesting of fertilized oocytes(s) or embryo(s) from one female (embryo donor) and transfer of those embryo(s) into another female (embryo recipient) whose reproductive status can be synchronized with that of the donor.

[0054] As used herein, the term “isolated” refers to molecular or biological or cellular material that is substantially free of other material (e.g., greater than 70%, or 80%, or 85%, or 90%, or 95%, or 98%). In one aspect, the term “isolated” refers to nucleic acids (e.g., DNA or RNA), miRNA, exosome or microvesicles, proteins or polypeptides, or cells or organelles, or tissues or organs, respectively, isolated from other DNA, RNA, miRNA, exosome or microvesicles, proteins or polypeptides, or cells or organelles, or tissues or organs, present in a natural source, which allow manipulation of the material to obtain results not achievable in its original or natural state, such as recombinant replication or mutation manipulations. The114899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)term “isolated” also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, “isolated nucleic acid” is intended to include nucleic acid fragments that do not occur in nature, and which do not occur in nature. The term “isolated” is also used herein to refer to polypeptides isolated from other cellular proteins, and is intended to include both purified and recombinant polypeptides, e.g., greater than 70%, or 80%, or 85%, or 90%, or 95%, or 98% pure. The term “isolated” is also used herein to refer to cells, exosomes or microvesicles, mirnas, or tissues isolated from other cells, exosomes or microvesicles, mirnas, or tissues, and is intended to encompass cultured and engineered cells or tissues and products produced or isolated therefrom.

[0055] As used herein, the term “edit” includes alterations in the nucleotide sequence of a polynucleotide, such as, for example, a gene, coding DNA sequence (CDS), or noncoding DNA sequence, compared to the wild-type sequence. The term “edits” may include insertions, deletions, splice-donor site edits, point-edits, and the like.

[0056] As used herein, the terms “gene-edited,” “genetically-edited,” and “genome-edited,” refer to the use of homing technology with naturally occurring or artificially engineered endonucleases, often referred to as “homing endonucleases,” or “targeting endonucleases.” “Genome editing” and “gene editing,” refer to altering the genome by deleting, inserting, or substituting specific nucleic acid sequences. The altering can be gene or location specific, but need not be altering the sequence of a gene per se. Genome editing can use endonucleases such as the CRISPR system to cut a nucleic acid, thereby generating a site for the alteration. Other endonucleases are available and are suitable for use; however, off-site cutting and specificity can be significant problems. In systems like CRISPR and others, the nuclease can be directed to the target site by complexing with a polynucleotide, herein called a “target sequence,” to introduce a site specific DSB. Not to be limited by theory, the DSB can then be repaired by endogenous non-homologous end joining (NHEJ) machinery. As used herein, “genotyping” refers to methods used to determine the genetic makeup of an organism, including but not limited to, conventional PCR, real-time PCR (qPCR), allele-specific PCR, and multiplex PCR. Restriction enzyme-based techniques like124899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)RFLP (Restriction Fragment Length Polymorphism) and CAPS (Cleaved Amplified Polymorphic Sequence) are also widely used. Hybridization-based methods such as microarrays (SNP arrays) and dot blot hybridization allow for detecting genetic variations. Sequencing methods like Sanger sequencing, next-generation sequencing (NGS), and wholegenome sequencing (WGS) are also key approaches, along with targeted sequencing. High-resolution melting (HRM) analysis, TaqMan assays, and molecular beacons are fluorescencebased genotyping techniques. Electrophoresis methods include SSCP (Single-Strand Conformation Polymorphism) and DGGE (Denaturing Gradient Gel Electrophoresis). Mass spectrometry-based techniques like MALDI-TOF (Matrix- Assisted Laser Desorption / Ionizati on-Time of Flight) are used for SNP genotyping, while CRISPR-based genotyping allows for targeted variant identification. Fragment analysis, digital droplet PCR (ddPCR), and eQTL (expression Quantitative Trait Locus) analysis are other valuable tools. Finally, comparative genomic hybridization (CGH) and LAMP (Loop-Mediated Isothermal Amplification) offer additional options for genotyping

[0057] The term “CRISPR” stands for “clustered regularly interspaced short palindromic repeats.” CRISPR systems include Type I, Type II, and Type III CRISPR systems. The term “Cas” refers to “CRISPR associated protein.” Cas proteins include but are not limited to Cas9 family member proteins, Cas6 family member proteins (e.g., Csy4 and Cas6), Cas5 family member proteins, and Cas 12 family member proteins. The term “Cas9” can generally refer to a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9% or 100% sequence identity and / or sequence similarity to a wild-type Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Illustrative Cas9 sequences are provided by SEQ ID NOs. 1-256 and 795-1346 of U.S. Patent Publication No.2016 / 0046963. SEQ ID NOs. 1-256 and 795-1346 of U.S. Patent Publication No.2016 / 0046963 are hereby incorporated herein by reference. “Cas9” can refer to can refer to a polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9%, 100% sequence identity and / or sequence similarity to a wild type Cas9 polypeptide (e.g., from S. pyogenes).

[0058] Cas9” can refer to the wild-type or a modified form of the Cas9 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, fusion,134899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)chimera, or any combination thereof. The term “Cas5” can generally refer to can refer to a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity and / or sequence similarity to a wild type illustrative Cas5 polypeptide (e.g., Cas5 from D. vulgaris). Illustrative Cas5 sequences are provided in Figure 42 of U.S. Patent Publication No. 2016 / 0046963. Figure 42 of U.S. Patent Publication No. 2016 / 0046963 is hereby incorporated herein by reference. “Cas5” can generally refer to can refer to a polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity and / or sequence similarity to a wild-type Cas5 polypeptide (e.g., a Cas5 from D. vulgaris). “Cas5” can refer to the wild- type or a modified form of the Cas5 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, fusion, chimera, or any combination thereof. The term “Cas6” can generally refer to can refer to a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity and / or sequence similarity to a wild type illustrative Cas6 polypeptide (e.g., a Cas6 from T. thermophilus). Illustrative Cas6 sequences are provided in Figure 41 of U.S. Patent Publication No. 2016 / 0046963. Figure 41 of U.S. Patent Publication No. 2016 / 0046963 is hereby incorporated herein by reference. “Cas6” can generally refer to can refer to a polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity and / or sequence similarity to a wild-type Cas6 polypeptide (e.g., from T. thermophilus). “Cas6” can refer to the wildtype or a modified form of the Cas6 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, fusion, chimera, or any combination thereof. The terms “CRISPR / Cas9” or “CRISPR / Cas9 system” refer to a programmable nuclease system for genetic editing that includes a Cas9 protein, or derivative thereof, and one or more non-coding guide RNAs (“gRNAs”) that provide the function of a CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA) for the Cas9. The crRNA and tracrRNA can be separate RNA molecules or can be combined into a single RNA molecule to produce a “single guide RNA” (sgRNA). The crRNA or the cRNA portion of the sgRNA provide sequence that is complementary to the genomic target.144899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)II. Embodiments

[0059] The following numbered embodiments also form part of the present disclosure:

[0060] 1. A method for producing a non-human gene-edited embryo with donor-derived germ cells, the method comprising:providing a host embryo comprising an inactivated PGC specification gene; and complementing the host embryo with donor cells to yield the gene-edited embryo.

[0061] 2. The method of embodiment 1, wherein the germ cells of the gene-edited embryo are exclusively derived from the donor, optionally wherein the donor cells are XX-chromosome-bearing cells.

[0062] 3. The method of embodiment 1 or 2, wherein the inactivated PGC specification gene is a NANOS gene.

[0063] 4. The method of embodiment 3, wherein the inactivated PGC specification gene is NANOS2.

[0064] 5. The method of any one of embodiments 1-4, wherein the host embryo is complemented at any stage from fertilized zygote to blastocyst stage.

[0065] 6. The method of any one of embodiments 1-4, wherein the host embryo is complemented at the morula stage as an early-stage embryo.

[0066] 7. The method of any one of embodiments 1-6, wherein the donor cells comprise one or more pluripotent cells.

[0067] 8. The method of embodiment 7, wherein the one or more pluripotent cells comprise embryonic stem cells or induced pluripotent stem cells.

[0068] 9. The method of any one of embodiments 1-8, wherein the non-human animal is a livestock animal.154899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0069] 10. The method of any one of embodiments 1-9, wherein the non-human animal is a bovine animal.

[0070] 11. The method of any one of embodiments 1-10, wherein the inactivated PGC specification gene is NANOS2, and wherein the NANOS2 is inactivated by introducing into the cell an engineered CRISPR / Cas system comprising a Cas protein or a polynucleotide encoding the Cas protein, and at least one guide RNA (gRNA) or a polynucleotide encoding the gRNA, wherein the gRNA specifically hybridizes to a NANOS2 sequence.[00711 12. The method of embodiment 11, wherein the at least one gRNA is selected from the group consisting SEQ ID NOs: 1-12.

[0072] 13. The method of embodiment 12, wherein the at least one gRNA comprises or consists of a gRNA pair.

[0073] 14. The method of embodiment 13, wherein the gRNA pair is selected from the group consisting of:(a) gRNA 370 (SEQ ID NO: 11) and gRNA 371 (SEQ ID NO: 12);(b) gRNA 360 (SEQ ID NO: 1) and gRNA 367 (SEQ ID NO: 8);(c) gRNA 361 (SEQ ID NO: 2) and gRNA 367 (SEQ ID NO: 8); and(d) gRNA 363 (SEQ ID NO: 4) and gRNA 368 (SEQ ID NO: 9).

[0074] 15. The method of any one of embodiments 11-14, wherein the Cas protein is selected from SpCas9 and OpenCRISPR-1 (OC-1).

[0075] 16. The method of embodiment 15, wherein the Cas protein is SpCas9.

[0076] 17. The method of embodiment 15, wherein the Cas protein is OC-1.

[0077] 18. A non-human gene-edited embryo comprising host cells and donor cells, wherein the host cells comprise an inactivated primordial germ cell (PGC) specification gene.164899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0078] 19. The non-human gene-edited embryo of embodiment 18, wherein the donor cells exclusively contribute to the germ cells of the non-human gene-edited embryo.

[0079] 20. A non-human gene-edited embryo comprising host cells and donor cells, wherein the host cells comprise an inactivated NANOS gene.

[0080] 21. The non-human gene-edited embryo of embodiment 20, wherein the donor cells exclusively contribute to the germ cells of the non-human gene-edited embryo.

[0081] 22. A non-human gene-edited embryo comprising host cells and donor cells, wherein the host cells comprise an inactivated NANOS2 gene.

[0082] 23. The non-human gene-edited embryo of embodiment 22, wherein the donor cells exclusively contribute to the germ cells of the non-human gene-edited embryo.

[0083] 24. A non-human gene-edited animal produced by the method of any one of embodiments 1-17, wherein the non-human gene-edited animal possesses a germline exclusively derived from the donor cells.

[0084] 25. A biological specimen obtained from the non-human gene-edited animal of embodiment 24.

[0085] 26. The method of any one of embodiments 1-17, further comprising creating a non-human gene-edited animal by implanting the gene-edited embryo into a surrogate mother and allowing the embryo to develop to term.

[0086] 27. The method of embodiment 26, wherein the non-human gene-edited animal is viable and fertile.

[0087] 28. A method for genotyping the non-human gene-edited embryo of any one of embodiments 18-24, the method comprising:obtaining a biological sample from the embryo;extracting nucleic acids from the sample; andperforming polymerase chain reaction (PCR) analysis to confirm the presence of the inactivated PGC specification gene and donor-derived genetic markers.174899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0088] 29. The method of embodiment 28, wherein the PCR analysis includes amplification of sequences flanking the inactivated PGC specification gene to verify the gene-editing event.

[0089] 30. The method of embodiment 28 or 29, wherein the PCR analysis includes real-time PCR to quantify the relative abundance of donor-derived genetic material.

[0090] 31. A method for genotyping the non-human gene-edited animal of embodiment 28, the method comprising:obtaining a biological specimen from the animal;extracting nucleic acids from the specimen; andperforming PCR analysis to confirm the donor-derived germline contribution.|0091] 32. The method of embodiment 31, wherein the PCR analysis includes amplification of sequences flanking the inactivated PGC specification gene to verify the gene-editing event.

[0092] 33. A method for producing a gene-edited bovine embryo with donor-derived bovine germ cells, the method comprising complementing a host embryo comprising an inactivated NANOS2 gene with XX-chromosome-bearing donor cells to yield the gene-edited bovine embryo, wherein the germ cells of the gene-edited embryo are exclusively derived from the donor.

[0093] 34. A gene-edited bovine embryo produced by the method of embodiment 33.

[0094] 35. A bovine animal produced by the method of embodiment 33.

[0095] 36. A biological specimen obtained from the bovine animal of embodiment 35.

[0096] In any of the embodiments described herein, a biological specimen can be or comprise any material originating from, derived from, or obtained from a living or previously living organism, including but not limited to whole organisms, body parts, cells, tissues, organs, and biological fluids. A biological specimen can include reproductive materials such184899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)as sperm, seminal fluid, ejaculate, ova, oocytes, embryos, and other gametes, as well as any fractions, isolates, derivatives, lysates, or preparations thereof. For example, as described herein, the biological specimen comprises a sperm cell sample obtained from a male of any of the non-human gene-edited animals of any of the embodiments described herein. As another example, as described herein, the biological specimen comprises an ejaculate obtained from a male of any of the non-human gene-edited animals of any of the embodiments described herein. In any of the embodiments described herein, the non-human gene-edited animal may be a bovine animal.III. Methods and Compositions

[0097] Inactivation of a primordial germ cell (PGC) specification gene, such as NANOS2 or NANOS3, leads to the loss of endogenous PGCs in a host embryo. When complemented with pluripotent cells from a desired donor, the resulting animal will have its germline entirely derived from the donor cells. This method allows the host embryo’s somatic cells to remain largely intact, ensuring that once the donor PGCs reach the gonads, robust spermatogenesis or oogenesis is established. This approach guarantees the preservation and amplification of valuable genetic traits through the next generation.

[0098] Unlike somatic cell nuclear transfer (SCNT) approaches, which often face challenges like low pregnancy success rates and epigenetic alterations, this technique offers a more reliable and scalable solution. It ensures that the majority of the resulting animal’s cells, excluding the germline, originate from the host embryo (with the PGC specification gene inactivated), resulting in the exclusive contribution of donor-derived germline cells. This unique method overcomes many of the drawbacks of SCNT while enabling efficient propagation of desired genetic traits for agricultural, conservation, or biotechnological applications.

[0099] Also, NANOS genes, particularly NANOS2 and NANOS3, are highly specific to germline cell differentiation and survival. As a result, NANOS genes are precise, controlling critical processes that are specific to the maintenance and differentiation of germline cells, particularly in the context of male germline development. This specificity provides a more targeted approach to germline modification, minimizing off-target effects194899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)and enhancing the efficiency of genetic interventions, especially in livestock species like cattle, pigs, and goats. The high conservation of NANOS genes across species further amplifies their utility, making them an ideal target for genetic engineering applications.

[0100] The present disclosure provides methods for producing a non-human gene-edited embryo or gene-edited animal with donor-derived pluripotent cells. The methods involve providing a host embryo with an inactivated primordial germ cell (PGC) specification gene, such as NANOS, and complementing the host embryo with donor cells to produce an embryo in which the germ cells are exclusively derived from the donor cells.

[0101] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the invention.Accordingly, the figures and detailed description are to be regarded as illustrative in nature and not restrictive.

[0102] The embryo complementation is performed in any early-stage embryo, iPSC, or stem cell host using genetic material from an early-stage embryo, iPSC, or stem cell donor. In the preferred embodiment, the gene-edited embryo is produced using blastocyst complementation. In another embodiment, the gene-edited embryo is produced using embryo-embryo aggregation techniques.

[0103] The embryo complementation is performed in an early-stage embryo, iPSC, or stem cell host using genetic material from an early-stage embryo, iPSC, or stem cell donor. In the preferred embodiment, the embryo complementation involves the complementation of blastomeres from a morula stage embryo into host male embryos (NANOS2 ) at the same developmental stage.

[0104] In another preferred embodiment, the embryo complementation involves the complementation of the inner cell mass (ICM) from an early-stage embryo into a host male embryo (NANOS2 ) at the same developmental stage.

[0105] In the preferred embodiment, the inactivation of the PGC specification gene may be accomplished by RNA-guided CRISPR-Cas9. In another embodiment, the204899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)inactivation of the PGC specification gene may be accomplished by use of a meganuclease. In another embodiment, the inactivation of the PGC specification gene may be accomplished by use of a TALEN. In another embodiment, the inactivation of the PGC specification gene may be accomplished by use of a zinc finger nuclease. In another embodiment, the inactivation of the PGC specification gene may be accomplished by use of base editors.

[0106] In an embodiment, the inactivated PGC specification gene comprises, but is not limited to, one or more of PRDM14, PRDM1, SALL4, IFITM1, DPP A3, TFAP2C, SOX15, c-kit (also known as KIT), DDX4, KITLG, DAZL, DND1, PRMT5, NANOG, AICDA, TIAL1, and NANOS. In a further embodiment, the inactivated PGC specification gene is NANOS2. In another embodiment, the inactivated PGC specification gene is NANOS3. In another embodiment, the inactivated PGC specification gene includes both NANOS2 and NANOS3. In another embodiment, the inactivated PGC specification gene is NANOSI.

[0107] The non-human animals can consist of mice, rabbits, and non-human primates as well as fish and livestock species, such as sheep, goats, and poultry. In the preferred embodiment, the non-human animals are ungulates. More specifically, in the preferred embodiment, the non-human animals are cattle (Bos taurus). In another embodiment, the non-human animals are pigs (Sus scrofa). These livestock species offer valuable applications in agricultural and biomedical fields due to their ability to produce large quantities of offspring and their suitability for genetic engineering.|0108] In a preferred embodiment, the donor cells comprise one or more cells from the inner cell mass. In another preferred embodiment, the donor cells comprise a blastomere. In another embodiment, the donor cells comprise embryonic stem cells. In another embodiment, the donor cells comprise embryonic germ cells. In another embodiment, the donor cells comprise induced pluripotent stem cells. In another embodiment, the donor cells comprise an embryo.

[0109] In some embodiments, the methods further comprise transferring the gene-edited embryo into a recipient female animal; and allowing the transferred gene-edited embryo to develop to term as a gene-edited animal. In some embodiments, the methods further comprise breeding the gene-edited animal with a second animal to produce one or214899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)more progeny animals. Non-human gene-edited embryos and gene-edited animals produced by the foregoing methods are provided. Also described herein is a non-human gene-edited embryo comprising host cells and donor cells. The host cells of the gene-edited embryo comprise an inactivated PGC specification gene and the donor cells exclusively contribute to the germ cells of the gene-edited embryo. In some embodiments, the inactivated PGC specification gene is NANOS. In another embodiment, the inactivated PGC specification gene is TFAP2C, SOX 15., and c-kit. Non-human gene-edited animals developed from the gene-edited embryos are also provided.

[0110] In some embodiments, the at least one gRNA is a pair of gRNAs. In some embodiments, the pair of gRNAs comprises two gRNAs selected from the group consisting of gRNA 360 (SEQ ID NO: 1), gRNA 361 (SEQ ID NO: 2), gRNA 362 (SEQ ID NO: 3), gRNA 363 (SEQ ID NO: 4), gRNA 364 (SEQ ID NO: 5), gRNA 365 (SEQ ID NO: 6), gRNA 366 (SEQ ID NO: 7), gRNA 367 (SEQ ID NO: 8), gRNA 368 (SEQ ID NO: 9), gRNA 369 (SEQ ID NO: 10), gRNA 370 (SEQ ID NO: 11), and gRNA 371 (SEQ ID NO: 12). In some embodiments, the pair of gRNAs comprises two gRNAs selected from the group consisting of gRNA 360 (SEQ ID NO: 1), gRNA 361 (SEQ ID NO: 2), gRNA 363 (SEQ ID NO: 4), gRNA 367 (SEQ ID NO: 8), gRNA 368 (SEQ ID NO: 9), gRNA 370 (SEQ ID NO: 11), and gRNA 371 (SEQ ID NO: 12).

[0111] In some embodiments, the pair of gRNAs comprises or consists of gRNA 370 (SEQ ID NO: 11) and gRNA 371 (SEQ ID NO: 12). In some embodiments, the pair of gRNAs comprises or consists of gRNA 360 (SEQ ID NO: 1) and gRNA 367 (SEQ ID NO: 8). In some embodiments, the pair of gRNAs comprises or consists of gRNA 361 (SEQ ID NO: 2) and gRNA 367 (SEQ ID NO: 8). In some embodiments, the pair of gRNAs comprises or consists of gRNA 363 (SEQ ID NO: 4) and gRNA 368 (SEQ ID NO: 9).

[0112] In a preferred embodiment, the at least one gRNA is a pair of gRNAs.Furthermore, in the preferred embodiment, the pair of gRNAs is sgRNA Pair #1 (gRNAs 370 (SEQ ID NO: 11) and 371 (SEQ ID NO: 12)).

[0113] In another embodiment, the method can further comprise breeding the gene-edited animal to a wild-type animal. In another embodiment, the method further comprises224899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)analyzing the genotype of the non-human gene-edited embryo to confirm the transmission of the gene edit. In yet another embodiment, the method further comprises creating a first-generation progeny from the gene-edited embryo. In another embodiment, the method further comprises genotyping the first progeny to confirm transmission of the gene edit. In yet another embodiment, the method further comprises creating a second-generation progeny either through natural or artificial processes. In another embodiment, the mate for the second progeny is selected based on desired characteristics. In another embodiment, the method further comprises mating the second-generation progeny with a selected mate to generate a third-generation progeny. This process of genetic selection and generation of progeny can be used to rapidly disseminate genes throughout a herd.

[0114] The present technology provides a method to generate a male (XY-chromosome-bearing) breeding animal that produces only X-chromosome-bearing sperm by combining targeted germline ablation in a male vector embryo with embryo complementation using XX blastomeres from a wild-type female donor embryo. In this approach, a male (XY) vector zygote is edited at the one-cell stage (e.g., using CRISPR nucleases) to knock out the NANOS2 gene, a gene required for the gonocyte-to-spermatogonia transition (GST) and maintenance of male germ cell lineage. This knockout ensures that endogenous germ cells of the male vector embryo cannot mature, thereby eliminating any capacity of the vector embryo’s own germline to contribute to spermatogenesis. At the morula stage (Day ~5 of culture), a defined number of XX blastomeres (e.g, 3-30 cells) from a wild-type female donor embryo are introduced into the NANOS2~'~ male vector embryo by micromanipulation to form a complemented chimeric embryo. The embryo is cultured up to Day ~7 and transferred to a recipient for gestation.

[0115] Following normal development, the resultant animal is somatically male (XY) and establishes a male testicular niche (e.g, Sertoli and Leydig lineages expressing Y-linked determinants) that supports male-pathway germ cell differentiation. Within this environment, the donor-derived XX germ cells undergo the canonical sequence: fetal primordial germ cell colonization, differentiation into gonocytes with neonatal arrest, GST at juvenile stages enabled by intact NANOS2 in the donor cells, and subsequent progression through spermatogenesis (spermatogonia — spermatocytes — spermatids — spermatozoa). Because234899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)the vector embryo’s endogenous germline is ablated and does not contribute any functional germ cells, all germ cells in the adult testis are donor-derived XX cells. Consequently, the ejaculate contains only X-bearing sperm, providing a biologically integrated route to female-biased progeny without external sperm sorting. Critically, the present technology produces a desired animal whose germline is exclusively XX and devoid of any germ cells derived from the male NANOS2~'~ vector embryo.

[0116] In some embodiments, donor-derived cells are female cells. In some embodiments, donor-derived cells are XX-chromosome-bearing cells.IV. Genome Editing SystemsCRISPR / Cas Systems

[0117] In some embodiments, the methods of the present technology relate to the use of a CRISPR / Cas system that binds to a target site in a region of interest in a genome, wherein the CRISPR / Cas system comprises a CRISPR / Cas nuclease and an engineered crRNA / tracrRNA (or single guide RNA (sgRNA) or guide RNA (gRNA)). In some embodiments, the CRISPR system generally comprises (i) a polynucleotide encoding a Cas protein, and (ii) at least one sgRNA for RNA-guided genome engineering.

[0118] Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Casl2a (also known as Cpfl), Csyl, Csy2, Cys3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Smrl, Cmr3, Cmr4, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. In some embodiments, the Cas protein is a Streptococcus pyogenes Cas9 protein. In some embodiments, the Cas protein is a Casl2a (Cpfl) protein. In some embodiments, the Cas protein is a Csml protein. These enzymes are known. For example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. The amino acid sequence of Francisella tularensis subsp. Novicida Cpfl protein may be found in the UniProt database under accession number A0Q7Q2. The amino acid sequence of Thermococcus onnurineus Csml protein may be found in the UniProt database under accession number B6YWB8.244899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0119] The sgRNA molecules comprise a crRNA-tacrRNA scaffold polynucleotide and a targeting sequence corresponding to a genomic target of interest.

[0120] In some embodiments, the CRISPR / Cas system recognizes a target site in a gene involved in germ cell development. In some embodiments, the CRISPR / Cas system recognizes a target in a PGC specification gene. In some embodiments, the CRISPR / Cas system recognizes a target in a NANOS2 gene. The CRISPR / Cas system as described herein may bind to and / or cleave the region of interest in a region upstream of the coding region of a gene involved in germ cell development. In some embodiments, the CRISPR / Cas system generates a specific sequence change in the 5’-UTR of a gene involved in germ cell development, such as generating a single nucleotide gene edit to form an out-of-frame start codon upstream of the gene’s ORF. In some embodiments, the gene edit (e.g., deletion, insertion, or substitution) results in production of an upstream, out-of-frame start codon that may result in the elimination of protein production or a nonfunctional protein. In some embodiments, the CRISPR / Cas system generates a specific sequence change or gene edit (e.g., insertion, deletion, or substitution) in the coding region or a non-coding region of a gene involved in germ cell development, such as generating a large deletion to form (1) an out-of-frame start codon upstream of the gene’s ORF, thereby suppressing expression of the gene involved in germ cell development, or (2) a non-functional protein product resulting from a frame shift downstream of the gene edit. In some embodiments, the large deletion is greater than 50 bases, greater than 100 bases, greater than 200 bases, greater than 500 bases, greater than 1000 bases, greater than 2000 bases, greater than 5000 bases, or greater than 10000 bases.

[0121] The CRISPR / Cas system can be based on the Cas9 nuclease and an engineered single guide RNA (sgRNA) that specifies the targeted nucleic acid sequence. Cas9 is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two non-coding RNAs:CRISPR RNA (crRNA) and trans-activating crRNA (tacrRNA).

[0122] The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt254899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can induce site-specific double strand breaks (DSBs) into genomic DNA of live cells. See, e.g., Mussolino, Nat. Biothechnol., 31:208-209 (2013). In some embodiments, the Cas9 protein is expressed in a cell as a fusion to a nuclear localization signal (NLS) to ensure delivery into nuclei. In some embodiments, the Cas9 protein is tagged (e.g., FLAG- or GFP-tagged). In some embodiments, promoters may be used to drive Cas9 expression in a cell. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes, or S. thermophiles Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme e.g., Cas9 enzyme) is codon-optimized for expression in a mammalian cell.

[0123] The CRISPR / Cas system can be based on the Cpfl nuclease and an engineered single guide RNA (sgRNA) that specifies the targeted nucleic acid sequence.

[0124] Cpfl is distinguished from Cas9 by its single RuvC endonuclease active site, its 5’ protospacer adjacent motif preference, and for creating sticky rather than blunt ends at the cut site. The Cpfl protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Cpfl does not have a HNH endonuclease domain, and the N-terminal of Cpfl does not have an alpha-helical recognition lobe, unlike Cas9. In some embodiments, the Cpfl protein is tagged e.g., FLAG- or GFP-tagged). In some embodiments, promoters may be used to drive Cpfl expression in a mammalian cell. In some embodiments, the Cpfl enzyme is Francisella tularensis subsp. Novicida Cpfl, and may include mutated Cpfl derived from these organisms. The enzyme may be a Cpfl homolog or ortholog. In some embodiments, the CRISPR enzyme e.g., Cpfl enzyme) is codon-optimized for expression in a mammalian cell.

[0125] The CRISPR / Cas system can be based on the Csml nuclease and an engineered single guide RNA (sgRNA) that specifies the targeted nucleic acid sequence.

[0126] Csml belongs to the CaslO family of endonucleases. Csml is the largest subunit of the Csm interference complex in the type III-A CRISPR system. Csml exhibits ssDNA-specific endo- and exonuclease activity. In some embodiments, promoters may be used to drive Csml expression in a mammalian cell. In some embodiments, the Csml264899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)enzyme is Thermococcus onnurineus Csml, and may include mutated Csml derived from these organisms. The enzyme may be a Csml homolog or ortholog. In some embodiments, the CRISPR enzyme (e.g., Csml enzyme) is codon-optimized for expression in a mammalian cell.

[0127] The CRISPR / Cas system can be based on the OpenCRISPR-1 (“OC-1”) nuclease and an engineered single guide RNA (sgRNA) that specifies the targeted nucleic acid sequence.

[0128] OC-1 is a synthetic nuclease belonging to a novel engineered family of CRISPR-associated (Cas) enzymes. OC-1 is the primary catalytic subunit of an interference complex designed to operate across multiple CRISPR system types. OC-1 exhibits doublestranded DNA-specific endonuclease activity with minimal off-target cleavage. In some embodiments, promoters may be used to drive OC-1 expression in a cell. The OC-1 enzyme is derived from rational protein engineering and may include variant forms optimized for enhanced specificity or activity. The enzyme may be an OC-1 homolog, ortholog, or further engineered derivative. In some embodiments, the CRISPR enzyme (e.g., OC-1 enzyme) is codon-optimized for expression in a mammalian cell.

[0129] The single guide RNA (sgRNA) is the second component of the CRISPR / Cas system that forms a complex with a Cas nuclease. The sgRNA is created by fusing crRNA with tacrRNA. The sgRNA guide sequence located at the 5’ end confers DNA target specificity. By modifying the guide sequence, sgRNAs with different target specificities can be designed to target any desired endogenous gene. In some embodiments, the target sequence is about 1,000, about 975, about 950, about 925, about 900, about 875, about 850, about 825, about 800, about 775, about 750, about 725, about 700, about 675, about 650, about 625, about 600, about 575, about 550, about 525, about 500, about 475, about 450, about 425, about 400, about 375, about 350, about 325, about 300, about 275, about 250, about 225, about 200, about 175, about 150, about 125, about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, or about 15 base pairs upstream of the transcription start site, or the target sequence may be any number of base pairs in-between these values upstream of the transcription start site. In some embodiments, the target sequence is about 1 to about 10 base pairs upstream of the transcription start site (e.g.,274899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)positions -10, -9, -8, -7, -6, -5, -4, -3, -2, or -1). In some embodiments, the target sequence is located within the open reading frame of the gene of interest. In some embodiments, the target sequence is located within a coding region of the gene of interest. In some embodiments, the CRISPR / Cas system comprises at least one sgRNA. In some embodiments, a target sequence of the at least one sgRNA is about 1,000, about 975, about 950, about 925, about 900, about 875, about 850, about 825, about 800, about 775, about 750, about 725, about 700, about 675, about 650, about 625, about 600, about 575, about 550, about 525, about 500, about 475, about 450, about 425, about 400, about 375, about 350, about 325, about 300, about 275, about 250, about 225, about 200, about 175, about 150, about 125, about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, or about 15 base pairs upstream of the transcription start site, or the target sequence may be any number of base pairs in-between these values upstream of the transcription start site. In some embodiments, the target sequence of the at least one sgRNA is about 1 to about 10 base pairs upstream of the transcription start site (e.g., positions -10, -9, -8, -7, -6, -5, -4, -3, -2, or -1). In some embodiments, the target sequence of the at least one sgRNA is located within the open reading frame of the gene of interest. In some embodiments, the target sequence of the at least one sgRNA is located within a coding region of the gene of interest. In some embodiments, the target sequences of the at least one sgRNA is located within the open reading frame of the gene of interest. In some embodiments, the target sequences of the at least one sgRNA is located within a coding region of the gene of interest. In some embodiments, the CRISPR / Cas system comprises one sgRNA, wherein the one sgRNA targets two regions of a gene having the same sequence, such as two inverted terminal repeats (ITRs). In some embodiments, the target sequences of the sgRNA are separated by at least 50 bases, at least 100 bases, at least 200 bases, at least 500 bases, at least 1000 bases, at least 2000 bases, at least 5000 bases, or at least 10000 bases.

[0130] In some embodiments, the CRISPR / Cas system comprises at least two sgRNAs. In some embodiments, a target sequence of at least one of the at least two sgRNAs is about 1,000, about 975, about 950, about 925, about 900, about 875, about 850, about 825, about 800, about 775, about 750, about 725, about 700, about 675, about 650, about 625, about 600, about 575, about 550, about 525, about 500, about 475, about 450, about 425, about 400, about 375, about 350, about 325, about 300, about 275, about 250, about 225,284899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)about 200, about 175, about 150, about 125, about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, or about 15 base pairs upstream of the transcription start site, or the target sequence may be any number of base pairs in-between these values upstream of the transcription start site. In some embodiments, the target sequence of at least one of the at least two sgRNAs is about 1 to about 10 base pairs upstream of the transcription start site (e.g., positions -10, -9, -8, -7, -6, -5, -4, -3, -2, or -1). In some embodiments, the target sequence of at least one of the at least two sgRNAs is located within the open reading frame of the gene of interest. In some embodiments, the target sequence of at least one of the at least two sgRNAs is located within a coding region of the gene of interest. In some embodiments, the target sequences of at least two of the at least two sgRNAs are located within the open reading frame of the gene of interest. In some embodiments, the target sequences of at least two of the at least two sgRNAs are located within a coding region of the gene of interest. In some embodiments, the CRISPR / Cas system comprises two sgRNAs, wherein the two sgRNAs have non-overlapping target sequences. In some embodiments, the target sequences of the two sgRNAs are separated by at least 50 bases, at least 100 bases, at least 200 bases, at least 500 bases, at least 1000 bases, at least 2000 bases, at least 5000 bases, or at least 10000 bases. In some embodiments, a gRNA comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-36. In some embodiments, a gRNA comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 1-12. In some embodiments, a gRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 1-36. In some embodiments, a gRNA comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 1-12.

[0131] It is not intended that the present technology be limited to any particular distance restraint with regard to the location of the guide RNA target sequence from the gene transcription start site. In some embodiments, the target sequence lies “in proximity to” a gene of interest, where “in proximity to” refers to any distance from the gene of interest, wherein the Cas-regulatory domain fusion is able to exert an effect on gene expression. In some embodiments, the target sequence lies upstream of the ORF of the gene of interest.294899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0132] The canonical length of the guide sequence is about 20 bp and the DNA target sequence is about 20 bp followed by a PAM sequence having the consensus NGG sequence. In some embodiments, sgRNAs are expressed in a mammalian cell using mammalian RNA polymerase III promoters.

[0133] When the DSBs are repaired by either NHEJ or HDR, the sequence at the repair site can be modified or new genetic information can be inserted (e.g., donor DNA comprising a desired gene edit can be inserted into the target gene at the break site).Although HDR typically occurs at lower and more variable frequencies than NHEJ, it can be leveraged to generate precise, defined modifications at a target locus in the presence of an exogenously introduced repair template. Accordingly, exogenous repair templates, designed by methods known in the art, can also be delivered into a cell, most often in the form of a synthetic, single-stranded DNA donor oligo or DNA donor plasmid, to generate a precise change in the genome. Single-stranded DNA donor oligos are delivered into a cell to insert or change short sequences (SNPs, amino acid substitutions, epitope tags, etc.) of DNA in the endogenous genomic target region. The benefits of using a synthetic DNA donor oligo is that no cloning is required to generate the donor template and DNA modifications can be added during synthesis for different applications, such as increased resistance to nucleases.Traditionally, the maximum insert length recommended for use with a DNA donor oligo is about 50 nucleotides.

[0134] In some embodiments, the present technology provides an engineered, programmable, non-naturally occurring CRISPR / Cas system comprising a Cas9 protein and one or more single guide RNAs (sgRNAs) that target the genomic loci of DNA molecules encoding one or more gene products associated with germ cell development, and the Cas9 protein cleaves the genomic loci of the DNA molecules encoding the one or more gene products, whereby expression of the one or more gene products is altered. In some embodiments, Cas9 introduces multiple DSBs in the same cell (i.e., multiplexes) via expression of one or more distinct guide RNAs.

[0135] In some embodiments, the present technology provides a method for targeted genomic modification of oocytes to alter the expression of at least one gene involved in germ cell development, the method comprising introducing into the cell an engineered304899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)CRISPR / Cas system comprising (a) an expression construct comprising a first polynucleotide encoding a Cas9 protein, or a variant thereof or a fusion protein therewith, and a second polynucleotide encoding a guide RNA comprising: (i) a crRNA-tracrRNA scaffold polynucleotide, and (ii) a targeting sequence operably linked to the crRNA-tracrRNA scaffold polynucleotide, where the targeting sequence corresponds to a genomic locus of interest, and (b) delivering the expression construct into the cell, where the first and second polynucleotides are expressed (transcribed) within the cell. This method can optionally further include visualizing, identifying, or selecting for cells having a genomic modification at the genomic locus of interest that is induced by the delivering the expression construct into the cell.

[0136] In some embodiments of the methods of the present technology, the Cas9 polypeptide and one or more guide RNA are encoded on a single vector. In some embodiments, the single vector is a plasmid. In some embodiments of the methods of the present technology, the Cas9 polypeptide and the one or more guide RNA are encoded on two separate vectors. In these methods, the steps generally follow the sequence of introducing into a cell containing and expressing a DNA molecule having a target sequence and encoding the gene involved in germ cell development an engineered CRISPR / Cas system comprising (a) a Cas9 polynucleotide or a conservative variant thereof, and a guide RNA comprising (i) a crRNA-tracrRNA scaffold polynucleotide, and (ii) a targeting sequence operably linked to the crRNA-tracrRNA scaffold polynucleotide, with the targeting sequence corresponding to a genomic locus of interest, and (b) delivering the two polynucleotides into the cell. In variations of this method, a donor polynucleotide having homology to the genomic target of interest is included in a co-transfection. In some variations of these methods, the transfected material can be either plasmid DNA or RNA generated by in vitro transcription. In still other variations, the methods for targeted genomic modification are multiplexed, meaning that more than one genomic locus is targeted for modification. In still other variations of these methods, the transformation of the cells can be followed by visualizing, identifying, or selecting for cells having a genomic modification at the genomic locus of interest.314899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)Meganucleases

[0137] In some embodiments, the compositions and methods described herein employ a meganuclease DNA binding domain for binding to a region of interest in the genome of a cell. Meganucleases are engineered versions of naturally occurring restriction enzymes that typically have extended DNA recognition sequences (e.g., about 14 to about 40 base pairs in length). Meganucleases (also known as homing endonucleases) are commonly grouped into five families based on sequence and structure motifs: the LAGLID ADG family (“LAGLID ADG”), the GIY-YIG family, the His-Cyst box family, the PD-(DZE)XK family, and the HNH family. In some embodiments, the meganuclease comprises an engineered homing endonuclease. The recognition sequences of homing endonucleases and meganucleases such as I-Sce, I-Ceul, PI-PspI, PI-5ce, I-5ceIV, I-CsmI, I-PanI, I-5ceII, I-Ppo\, I-5ceIII, I-Crel, I-TevI, I-TevII, and LTevIII are known.

[0138] In some embodiments, the meganuclease is tailored to recognize a target in a PGC specification gene. In some embodiments, the meganuclease is tailored to recognize a target in a NANOS2 gene. The meganucleases as described herein may bind to and / or cleave the region of interest in a region upstream of the coding region of a gene involved in germ cell development. Gene insertion or correction can be achieved by the introduction of a DNA repair matrix containing sequences homologous to the endogenous sequence surrounding the DNA break. Gene edits can be created either at or distal to the break. In some embodiments, the meganuclease generates a specific sequence change in the 5’-UTR of a gene involved in germ cell development, such as generating a single nucleotide gene edit to form an out-of-frame start codon upstream of the gene’s ORF.TALENs

[0139] In some embodiments, the compositions and methods described herein employ transcription activator-like effector nucleases (TALENs) to edit genomes by inducing doublestrand breaks (DSBs). TALENs are restriction enzymes that can be engineered to cleave specific sequences of DNA. TALENs are constructed by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (e.g., a nuclease domain such as that derived from the FokI endonuclease). Transcription activator-like effectors (TALEs) can be324899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)engineered according to methods known in the art to bind to a desired DNA sequence, and when combined with a nuclease, provide a technique for cutting DNA at specific locations. For example, after a target sequence in a gene involved in germ cell development is identified, a corresponding TALEN sequence is engineered and inserted into a plasmid. The plasmid is inserted into a target cell where it is translated to produce a functional TALEN, which then enters the nucleus where it binds to and cleaves its target sequence. Such an approach can be employed to introduce an exogenous DNA sequence into the target gene as the DSB is being repaired through either homology-directed repair or non-homologous endjoining. For example, in some embodiments, the use of TALEN technology generates a specific sequence change (e.g., insertion, deletion, or substitution) in the 5’-UTR of a gene involved in germ cell development, resulting in the production of an out-of-frame start codon upstream of the gene’s ORF.ZFNs[01401 In some embodiments, the compositions and methods described herein employ zinc finger nucleases (ZFNs) to edit genomes by inducing double-strand breaks (DSBs). ZFNs are artificial restriction enzymes generated by fusing a zinc finder DNA-binding domain to a DNA cleavage domain (e.g., a nuclease domain such as that derived from the FokI endonuclease). ZFNs can be engineered to bind and cleave DNA at specific locations. ZFNs contain two protein domains. The first domain is the DNA-binding domain, which contains eukaryotic transcription factors and the zinc finger. The second domain is a nuclease domain that contains the FokI restriction enzyme responsible for cleaving DNA. ZFNs can be engineered according to methods known in the art to bind to a desired DNA sequence and cleave DNA at specific locations. For example, after a target sequence in a gene involved in germ cell development is identified, a corresponding ZFN sequence is engineered and inserted into a plasmid. The plasmid is inserted into a target cell where it is translated to produce a functional ZFN, which then enters the nucleus where it binds to and cleaves its target sequence introducing a double strand break (DSB). Such an approach can be employed to introduce an exogenous DNA sequence into the target gene as the DSB is being repaired through either homology-directed repair or non-homologous end-joining. For example, in some embodiments, the use of ZFN technology generates a specific sequence334899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)change in the 5’-UTR of a gene involved in germ cell development, such as the insertion of an out-of-frame start codon upstream of the gene’s ORF.EXAMPLES

[0141] The present teachings include descriptions provided in the Examples that are not intended to limit the scope of any claim or embodiment. The following non-limiting examples are provided to further illustrate the present teachings. Those of skill in the art. in light of the present disclosure, will appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.Example 1 : Producing IVF embryos (as Donor or Host Embryo)

[0142] The present Example describes methods for genetically editing a host embryo for use in accordance with the present technology.

[0143] In the following experiments, the host embryo was edited, and the donor embryo was not edited. The host embryo was a bovine Holstein oocyte.

[0144] The donor embryo could also be an in vivo derived embryo that is flushed from a female at the appropriate embryonic stage. In cattle, this can be accomplished non-surgically 4-7 days after breeding.

[0145] The donor embryo can also be generated from a cell line via nuclear transfer methods.

[0146] Oocytes were purchased as cumulus-oocyte complexes (COC’s) from ART (Monona, WI) in Maturation Media. After oocyte maturation, COCs were washed twice through holding media, and groups of 10 COCs in 10 pl of holding media were placed into 35 pl drops of IVF media containing 33 pg of heparin / ml and 180 ng of epinephrine / ml. Holstein semen was thawed at 37°C for 60 sec then overlaid on a 45% / 90% Percoll gradient, 450 pl of each. Semen was centrifuged for 10 min at 400 x g, then the supernatant was removed, and sperm were washed with 1 ml of holding media. Sperm were centrifuged for 5 min at 100 x g, then 30 pl of pellet was removed and placed into a separate tube. Sperm were344899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)counted on a Neubauer chamber and diluted with IVF media to achieve a concentration of 1 million sperm / ml in the IVF drop, adding 5 pl of the sperm suspension to each drop. Sperm and COCs were co-incubated for 16-18 hrs at 38.5°C at 6% CO2 and atmospheric air.Example 2: Inner cell mass (ICM) embryo complementation[01471 For ICM embryo complementation experiments, blastocyst stage donor and host embryos were utilized. The donor blastocyst has the ICM excised from the embryo using an embryo splitting blade attached to a micromanipulator. Using the micromanipulator, the blade was brought down in the z-axis from above the embryo to press down on the blastocyst, resulting in slicing of the embryo. This enabled the ICM specifically to be cut away from the blastocyst. After the ICM was cut away from the blastocyst, the isolated ICM was cut into 2 or 3 pieces so that they were small enough to insert into the host blastocyst using micromanipulation tools designed for injection. Injection into the host blastocyst was assisted by a hatching laser and the ICM piece was inserted into the blastocoel cavity. After ICM injection, the injection tool was removed and the host embryo collapsed, resulting in a host embryo that contains the donor ICM.Example 3: Successful incorporation of complemented donor cells

[0148] Following embryo complementation from either ICM or blastomere donor cells, resulting embryos were transferred into estrus synchronized surrogate cattle. At roughly 90 days of gestation, fetuses were recovered and the developing gonad was observed. As shown in FIG. 3, incorporation of donor cells — which expressed fluorescent proteins for labeling purposes — into fetal testes was observed. FIG. 4 is a formalin fixed cross section of a fetal testis highlighting presence of Green fluorescent protein (GFP)-expressing donor cells present in the seminiferous tubules. FIG. 5 is a formalin fixed cross section of a fetal testis highlighting presence of Red fluorescent protein (RFP)-expressing donor cells present in the seminiferous tubules.Example 4: Design of guide RNAs (gRNA) for gene editing of host embryos

[0149] This example illustrates the design of gRNAs for the deletion of NANOS2 using nucleases with NGG PAMs (Protospacer Adjacent Motif).354899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0150] gRNAs were designed to be used with Cas9-like or other nucleases that require an NGG PAM. Cas9 typically uses a 5’-NGG-3’ PAM sequence (where N is A, T, C, or G) that is located downstream (3’ end) of the target DNA sequence (spacer). gRNAs were designed 150 bp upstream and 150 bp downstream of the NANOS2 exon with 5’-NGG-3’ PAMs and a 20 bp spacer sequence (Table 1 below).Table 1: NGG Guide RNAs

[0151] Additional gRNAs were designed to be used with Cas9-like or other nucleases that require a non-NGG PAM. In particular, gRNAs were designed to be used with Casl2a-like or other nucleases that require a non-NGG PAM. Casl2a typically uses a 5’-TTTV-3’ PAM sequence (where V is A, C, or G) that is located upstream (5’ end) of the target DNA sequence (spacer). gRNAs were designed 150 bp upstream and 150 bp downstream of the NANOS2 exon with 5’-TTTV-3’ PAMs and a 24 bp spacer sequence (Table 2 below). Table 3 and Table 4 show exemplary CCCA guides and other non-NGG guides, respectively.Spacer sequences can be between 20 and 24 bp.

[0152] Although, a 5’-TTTV-3’ PAM sequence is often preferred for these nucleases they can also have variation in PAM specificity. For example, gRNAs were designed 150 bp upstream and 150 bp downstream of the NANOS2 exon with 5’-CCCA-3’ PAMs and a 24 bp spacer sequence (Table 3 below). This is just one example of a variant PAM that was tested.364899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)Table 2: Exemplary TTTV GuidesTable 3: Exemplary CCCA GuidesTable 4: Exemplary non-NGG guides4899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)Example 5: Transfection of bovine embryonic fibroblasts to produce gene-edited cells

[0153] In this Example, nucleofection for delivering guide RNA / Cas9 endonuclease is described. To test the DNA cutting activity in living cells to produce an edited bovine NANOS2 allele, the CRISPR-Cas endonuclease and guide RNA combinations listed in Table 1 were nucleofected into bovine fetal fibroblast cells prepared from 55-65 days post conception fetuses. The ribonucleoprotein (RNP) complex was generated by the addition of 3.2 pg (20 pmol) of Cas9 protein (5. pyogenes or S. thermophilus) or OpenCRISPR-1 and 2.2 pg (60 pmol) each of in vitro synthesized guide RNA and combined in water to a total volume of 2.23 pl (see Example 8 below). Then, the RNP mixture was nucleofected into bovine fetal fibroblast (BEF) cells using a Lonza electroporator. In preparation for nucleofection, BEF cells were harvested using TrypLE express (recombinant Trypsin), upon which the culture medium is removed from cells, washed 1 X with HBSS or DPBS, and incubated for 3 - 5 minutes at 37.0 - 38.5°C in the presence of TrypLE. Cells were then harvested with complete medium. Cells were pelleted via centrifugation (300g x 5 min @ RT), supernatant was discarded, and then the cells were resuspended in 10 mL PBS to obtain single cell suspension counting cells using Trypan blue staining.

[0154] Cells were re-pelleted via centrifugation, the supernatant was discarded, and the cells were resuspended in nucleofection buffer Pl at a final concentration of 7.5xl06cells / ml. 20 pl of the cell suspension was added to each well of a nucleofection tube containing the RNP mixture using a multichannel pipette, then mixed gently to resuspend the cells. The RNP / cell mixture was transferred into the nucleofection tube, nucleofected with program CM137, and 80 pl of warm Embryonic Fibroblast Medium, EFM, (Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2.77 mM glucose, 1.99 mM L-glutamine, and 0.5 mM sodium pyruvate, supplemented with 100 pM 2-Mercaptoethanol, IX Eagle’s minimum essential medium non- essential amino acids (MEMNEAA), 100 pg / mL Penicillin-Streptomycin, and 12% Fetal Bovine Serum was added to each well after nucleofection. The suspensions were mixed gently by pipetting, and then 100 pl were transferred to a 12 well plate containing 900 pl of EFM pre-incubated at 38.5°C. The plate was then incubated at 38.5°C, 5% CO2 for 48 hours. Forty-eight hours post nucleofection, genomic DNA was prepared from transfected and control BFF cells, 15 pl of384899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)QUICKEXTRACT™ DNA Extraction Solution were added to pelleted cells, the cells were then lysed by incubating for 10 mins at 37°C, for 8 mins at 65°C, for 5 mins at 95°C, andthen lysate was held at 4°C until used for DNA sequencing.

[0155] For short sequence reads, two-step PCR was used to amplify and sequence the region of interest. The first step was a locus-specific PCR which amplified the locus ofinterest from the DNA sample using a combined locus-specific primer with a vendor-specific primer. The second step attached the sequencing index and adaptor sequences to theamplicon from the first step so that sequencing could occur.

[0156] The locus-specific primers for the first step PCR were chosen so that they amplified a region <300 bp such that ILLUMINA® paired-end sequencing reads could span the amplified fragment. Multiple amplicons were preferred to provide redundancy should deletions or naturally occurring point mutations prevent primers from correctly binding.Sequence data for the amplicon was generated using an ILLUMINA® sequencing platform (MISEQ®, ILLUMINA®, San Diego, Calif.). Sequence reads are analyzed to characterize the outcome of the editing process.Table 5. Detection of NAN0S2 deletions in tested embryos394899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0157] Accordingly, these results demonstrate that the methods of the present technology are effective for introducing deletions in a NAN0S2 gene in an embryo.Example 6: Zygote Mi croini ection to Produce Gene-Edited Embryos

[0158] Dual guide sgRNP’s were assembled and mixed as follows: IDT Alt-R Cas9 V3 Nuclease (100 pg = 610 pmol; 10 pg / pl = 61 pmol) or OpenCRISPR-1 protein; sgRNA stock concentration: 1.5 pg / pl; Each RNP Complex Concentration: 5.0 pg of Cas9 protein + 3.42 pg of each sgRNA. sgRNAs were incubated @ 95°C for 2 min in thermal cycler, briefly spun down and then allowed to cool to room temperature. The H2O and Cas9 protein were mixed by pipetting up and down, and were then kept on ice. The denatured sgRNAs were then added to the Cas9 / H2O mixture. The mixture was briefly mixed by pipetting, and then quickly centrifuged if necessary. The mixture was then incubated for 10 min at room temperature.

[0159] RNP complex from Table 6 was diluted with 193.0 pl of H2O to 7.0 pl of the combined RNP complexes. The working concentration was 50.2 ng / pl of Cas9 and 17.2 ng / pl of each sgRNA.404899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)Table 6. RNP Complex

[0160] The sgRNP solution was injected into the cytoplasm of presumptive zygotes at 16-17 hours post-fertilization by using a single pulse from a FemtoJet 4i microinjector (Eppendorf; Hamburg, Germany) with settings at pi=200 hPa, ti=0.25 s, pc=15 hPa. Glass capillary pipettes with an outer diameter of 1.2 mm and an inner diameter of 0.94 mm were pulled to a very fine point of < 0.5 pm (Sutter Instrument, Navato, CA, USA).Microinjection was performed in TL-Hepes (ABT360, LLC) supplemented with 3 mg / ml BSA (Proliant) on the heated stage of an inverted microscope equipped with Narishige (Narishige International USA, Amityville, NY) micromanipulators. Following injections, presumptive zygotes were cultured for 4-7 days in BO-IVC (IVF Bioscience, Falmouth, Cornwall, UK) in an incubator environment of 5% CO2, 5% O2, 90% N2.

[0161] The embryo complementation method utilized blastomeres from an early stage embryo, for example a morula stage, or inner cell mass (ICM) cells from an early stage embryo such as a blastocyst stage embryo. To complement with blastomeres from a morula stage embryo, several blastomeres from the donor embryo were aspirated into a glass aspiration tool. Then with the same tool, some of the blastomeres were injected into the host morula stage embryo, making sure the donor blastomeres are injected into the center region of the host embryo. ICM complementation was performed as described in Example 2.

[0162] The NANOS2 knock-out efficiency for four sgRNA pairs is depicted in Table 7. An exemplary NANOS2 knock-out repair outcome following use of sgRNA Pair #1 (370 and 371) is displayed in FIG. 7.

[0163] These results demonstrate that particular pairs of gRNAs are effective for introducing gene-edited embryos containing a deletion of the NANOS2 gene.414899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)Table 7. NAN0S2 Knock-out efficiency in embryos# analyzed refers to the number of embryos analyzed for each treatment condition. #edited refers to number of embryos which demonstrated any editing at the NAN0S2 locus, regardless of editing type or effect. # of embryos with gene deletion / no WT refers to the number of embryos which had 1 or more alleles which resulted in the deletion of NAN0S2 guide sequence, with up to 15bp remaining between the cutsites, with or without chewback from the cutsite, and no sequence corresponding to the WT sequence. Avg cumulative gene deletion frequency corresponds to those embryos in the line above (#of embryos with gene deletion alleles / no WT); cumulative frequency of deletion alleles refers to the sum of the frequencies (i.e. number of reads out of total reads that correspond to the particular allele) for all gene deletion alleles, as described above; the average cumulative frequency is the average across all of those embryos. Avg number of different gene deletion alleles is the average number of gene deletion alleles that correspond to the cumulative gene deletion frequency.

[0164] The complementation efficiency of the complementation method disclosed in FIG. 1 can be read in Table 8 and visualized in FIGs.2-3.Table 8. Complementation efficiency424899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)Example 7: In vivo gene editing by injection of RNPs

[0165] For each experiment, the gRNAs were generated by in vitro transcription or chemically synthesized (synthetic sgRNA) by a commercial vendor (IDT) and complexed with IDT Alt-R spCas9 V3 in water, using 3.2 pg of Cas9 protein and 2.2 pg of gRNA in a total volume of 2.23 pl. For dual guide experiments the resulting RNP complexes were then combined 1:1 in a total volume of 2.23 pl to generate gRNA pairs. For both single and dual guide experiments, the end result was a solution of about 20 pmol Cas9 and 60 pmol sgRNAs. As used throughout these examples, this is the lx solution, and contains about 1.5 amol, or about 890,000 copies of the RNP complex. This solution was then diluted to make other concentrations of RNP complex: a 1 :2 dilution (l / 2x or 0.5x), which results in injection of about 0.75 amol, or about 445,000 copies of the RNP complex, a 1 :4 dilution (l / 4x or 0.25x), which results in injection of about 0.375 amol, or about 222,500 copies of RNP complex, and a 1:8 dilution (l / 8x or 0.125x), which results in injection of about 0.1875 amol, or about 111,250 copies of the RNP complex.|0166] The sgRNP solution was injected into the cytoplasm of presumptive zygotes at 16- 17 hours post-fertilization by using a single pulse from a FEMTOJET® 4i microinjector (Eppendorf; Hamburg, Germany), which released about 20 picoliters per zygote.

[0167] Microinjection was performed in TL-Hepes (ABT3 0, LLC) supplemented with 3 mg / ml BSA (Proliant) on the heated stage of an inverted microscope equipped with Narishige micromanipulators (Narishige International USA, Amityville, NY). Following injections, presumptive bovine zygotes were cultured for 7 days in PZM5 (Cosmo Bio, Co LTD, Tokyo, Japan) or a bovine in vitro culture medium in an incubator environment of 5% CO2, 5% O2, 90% N2.Example 8, Exemplary repair outcomes

[0168] To assess the efficacy of the NANOS2 knock-out using various pairs of gRNAs, samples containing genomic DNA from gene-edited cells were subjected to genomic sequencing. Exemplary repair outcomes resulting from attempted large deletions within and around the NANOS2 gene are summarized in Tables 9-12, below. The 5’ sequence represents from the forward sequencing primer to the cutsite for each guide; the center 434899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)sequence represents between the cutsites; and the 3’ sequence represents (5’ to 3’) from the cutsite to the reverse primer. By these definitions, Applicant observed functional deletion when there is no sequence present in the center column, or no sequence that would encode a functional NANOS2 protein.(0169] These results demonstrate that the present technology is capable of generating large deletions in a NANOS2 gene in an embryo, such deletions resulting in a variety of repair outcomes. “Ct.” refers to the raw count of cells having the listed repair outcome. “Fr.” refers to the frequency of cells having the listed repair outcome.Table 9. Repair Outcomes for Guide Pair 360 / 367444899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)454899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)Table 10. Repair Outcomes for Guide Pair 361 / 367464899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)474899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)484899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)Table 11. Repair Outcomes for Guide Pair 363 / 368494899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)504899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)Table 12. Repair Outcomes for Guide Pair 370 / 371514899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)524899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)Example 9, Assessment of NANOS2 vector embryos|0170] NANOS2 -I- status was determined by genomic sequencing. Genomic DNA was extracted from blood and / or ear fragments. Sequencing was performed as described above. Frequency of each allele and description are represented in the first table. Allele sequences for each animal are shown in Table 13.Table 13. Assessment of calf NANOS2 status534899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0171] Post-edit NANOS2 deletion repair outcomes for each of the calves is shown in Table 14Table 14. NAN0S2 deletion repair outcomes for select calves544899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)

[0172] As shown in FIG. 8, chimeric bulls having testes that were positive for RFP-positive cells, which are bright white in FIG. 8 and are encircled with a dotted white line, were successfully generated. At 5 months, two chimeric bulls, Calf 30 and Calf 32, displayed RFP (red fluorescent protein, which appears as bright white) incorporation in the testes.These chimeric animals were generated by complementing a.NANOS2~'~ male vector embryo (represented in grey) with six blastomeres derived from a cloned female donor embryo expressing RFP (represented in red, which appears as light gray cells encircled by dotted black lines in the schematic images of FIG. 8). The images reveal distinct red donor cells in 5 month postnatal testis. These observations confirm the persistence and integration of XX donor cells in the gonadal tissue of both calves well after birth, demonstrating successful long-term incorporation of female donor cells in the male reproductive system.

[0173] These results demonstrate that the present technology is effective in generating chimeric bulls having germ cells derived from a XX donor cell. Such bulls express XX-bearing cells in the testes.EQUIVALENTS

[0174] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this present technology is not554899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0175] Each and every publication and patent mentioned in the above specification is herein incorporated by reference in its entirety for all purposes. Various modifications and variations of the described methods and system of the present technology will be apparent to those skilled in the art without departing from the scope and spirit of the present technology. Although the present technology has been described in connection with specific embodiments, the present technology as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the present technology which are obvious to those skilled in the art and in fields related thereto are intended to be within the scope of the following claims.564899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)SEQUENCES574899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)584899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)594899-1857-1399.1

Claims

Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)WHAT IS CLAIMED IS:

1. A method for producing a non-human gene-edited embryo with donor-derived germ cells, the method comprising:providing a host embryo comprising an inactivated PGC specification gene; andcomplementing the host embryo with donor cells to yield the gene-edited embryo, wherein the germ cells of the gene-edited embryo are exclusively derived from the donor.

2. The method of claim 1, wherein the inactivated PGC specification gene is NANOS.

3. The method of claim 1, wherein the inactivated PGC specification gene is NANOS2.

4. The method of claim 1, wherein the host embryo is complemented at any stage from fertilized zygote to blastocyst stage.

5. The method of claim 1, wherein the host embryo is complemented at the morula stage as an early-stage embryo.

6. The method of claim 1, wherein the donor cells comprise one or more pluripotent cells.

7. The method of claim 6, wherein the one or more pluripotent cells comprise embryonic stem cells or induced pluripotent stem cells.

8. The method of claim 1, wherein the non-human animal is a livestock animal.

9. The method of any one of claims 1-8, wherein the non-human animal is a bovine animal.

10. The method of any one of claims 1-9, wherein the inactivated PGC specification gene is NANOS2, and wherein the NANOS2 is inactivated by introducing into the cell an engineered CRISPR / Cas system comprising a Cas protein or a polynucleotide 604899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)encoding the Cas protein, and at least one guide RNA (gRNA) or a polynucleotide encoding the gRNA, wherein the gRNA specifically hybridizes to a NANOS2 sequence.

11. The method of claim 10, wherein the at least one gRNA is selected from the group consisting SEQ ID NOs: 1-12.

12. The method of claim 11, wherein the at least one gRNA comprises or consists of a gRNA pair.

13. The method of claim 12, wherein the gRNA pair is selected from the group consisting of(a) gRNA 370 (SEQ ID NO: 11) and gRNA 371 (SEQ ID NO: 12); (b) gRNA 360 (SEQ ID NO: 1) and gRNA 367 (SEQ ID NO: 8);(c) gRNA 361 (SEQ ID NO: 2) and gRNA 367 (SEQ ID NO: 8); and (d) gRNA 363 (SEQ ID NO: 4) and gRNA 368 (SEQ ID NO: 9).

14. The method of any one of claims 10-13, wherein the Cas protein is selected from SpCas9 and OpenCRISPR-1 (OC-1).

15. The method of claim 14, wherein the Cas protein is SpCas9.

16. The method of claim 14, wherein the Cas protein is OC-1.

17. The method of any one of claims 1-16, wherein the donor cells are XX-chromosome-bearing cells.

18. A non-human gene-edited embryo comprising host cells and donor cells, wherein the host cells comprise an inactivated primordial germ cell (PGC) specification gene, and wherein the donor cells exclusively contribute to the germ cells of the non-human gene-edited embryo.

19. A non-human gene-edited embryo comprising host cells and donor cells, wherein the host cells comprise an inactivated NANOS gene, and wherein the donor cells exclusively contribute to the germ cells of the non-human gene-edited embryo.614899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)20. A non-human gene-edited embryo comprising host cells and donor cells, wherein the host cells comprise an inactivated NANOS2 gene, and wherein the donor cells exclusively contribute to the germ cells of the non-human gene-edited embryo.

21. A non-human gene-edited animal produced by the method of any one of claims 1-16, wherein the non-human gene-edited animal possesses a germline exclusively derived from the donor cells.

22. A biological specimen obtained from the non-human gene-edited animal of claim 20.

23. The method of claim 1, further comprising creating a non-human gene-edited animal by implanting the gene-edited embryo into a surrogate mother and allowing the embryo to develop to term.

24. The method of claim 23, wherein the non-human gene-edited animal is viable and fertile.

25. A method for genotyping the non-human gene-edited embryo of claim 20, the method comprising:obtaining a biological sample from the embryo;extracting nucleic acids from the sample; andperforming polymerase chain reaction (PCR) analysis to confirm the presence of the inactivated PGC specification gene and donor-derived genetic markers.

26. The method of claim 25, wherein the PCR analysis includes amplification of sequences flanking the inactivated PGC specification gene to verify the gene-editing event.

27. The method of claim 25, wherein the PCR analysis includes real-time PCR to quantify the relative abundance of donor-derived genetic material.

28. A method for genotyping the non-human gene-edited animal of claim 21, the method comprising:obtaining a biological specimen from the animal;extracting nucleic acids from the specimen; and624899-1857-1399.1Atty. Dkt. No.: 102726-0302(TD-36-2025-WO1)performing PCR analysis to confirm the donor-derived germline contribution.

29. The method of claim 28, wherein the PCR analysis includes amplification of sequences flanking the inactivated PGC specification gene to verify the gene-editing event.

30. A method for producing a gene-edited bovine embryo with donor-derived bovine germ cells, the method comprising complementing a host embryo comprising an inactivated NANOS2 gene with XX-chromosome-bearing donor cells to yield the gene-edited bovine embryo, wherein the germ cells of the gene-edited embryo are exclusively derived from the donor.

31. A gene-edited bovine embryo produced by the method of claim 30.

32. A bovine animal produced by the method of claim 30.

33. A biological specimen obtained from the bovine animal of claim 32.634899-1857-1399.1

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