Transgenic animals and recombinant host cells comprising direwolf specific gene variants and methods of creating same

Abstract

Provided herein are isolated nucleic acids, vectors, recombinant cells, and transgenic animals capable of expressing at least one direwolf (Aenocyon dirus) gene variant associated with coat composition and / or vibrissae. Also provided are methods of making recombinant cells and / or transgenic animals that comprise at least one direwolf (Aenocyon dirus) gene variant associated with coat composition and / or vibrissae.

Description

Attorney Docket No.: 069296.11210 / 28WO2TRANSGENIC ANIMALS AND RECOMBINANT HOST CELLS COMPRISING DIREWOLF SPECIFIC GENE VARIANTS AND METHODS OF CREATING SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 746,804, filed on January 17, 2025, and U.S. Provisional Application No. 63 / 746,822, filed on January 17, 2025. Each disclosure is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The present disclosure generally relates to gene edited, and / or reprogrammed mammalian cells and uses thereof and methods of making the same. In particular, the disclosure relates to gene edited, and / or reprogrammed mammalian cells containing direwolf (Aenocyon diru ) gene variants and methods of making the same.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] This application contains a sequence listing, which is submitted electronically. The contents of the electronic sequence listing (069296.28WO2 Sequence Lisitng.xml; size:14,819,662 bytes; and creation date of January 7, 2026) is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0004] The generation of direwolves and the restoration of a population of this extinct species can have a wide array of benefits, including, but not limited to, conservation and de-extinction efforts, as the methods used herein can functionally restore an extinct species and preserve species diversity to combat climate change effects. Without being bound by theory, it is thought that restoration of the direwolf could contribute to restoring ecosystems, since the role of apex predators in maintaining the balance in ecosystems is well established. Additionally, as part of the genomic editing methods described herein, novel genomic information about living gray wolf species, as well as the extinct direwolf has been produced. This information can aid in the research efforts around evolutionary biology, speciation, and ecology. Finally, the de-extinction of the direwolf will advance synthetic biology and will serve as a precedent for advanced multiplexed editing in organisms, as well as providing a tool-kit for species preservation of other extinct or near extinct species.4908-6027-7382.1Attorney Docket No.: 069296.11210 / 28WO2

[0005] Accordingly, there is a need to restore direwolves. This disclosure provides methods to generate recombinant host cells and transgenic animals with phenotypes comprising the coat composition of direwolves and / or the vibrissae of direwolves.BRIEF SUMMARY OF THE INVENTION

[0006] Provided herein are transgenic animals comprising at least one direwolf (Aenocyon dirus) gene variant associated with (a) coat composition; and / or (b) vibrissae.

[0007] In certain embodiments, the at least one direwolf (Aenocyon dirus) gene variant associated with coat composition is selected from the group consisting of a Beta-defensin 103 (CBD103) variant, a Fibroblast growth factor 5 (FGF5) variant, a Melanocortin receptor 1 (MC1R) variant, a Major facilitator superfamily domain containing 12 (MFSD12) variant, an Agouti signaling protein (ASIP) variant, a Serine peptidase (CORIN) variant, a Dedicator of cytokinesis 7 (DOCK7) variant, a Keratin 71 (KRT71) variant, a Keratin 76 (KRT76) variant, a Melanocyte inducing transcription factor (MITF) variant, a Melanophilin (MLPH) variant, a Melanoregulin (MREG) variant, and a Tyrosinase related protein 1 (TYRP1) variant. In certain embodiments, the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.

[0008] In certain embodiments, the at least one direwolf (Aenocyon dirus) gene variant associated with vibrissae is selected from a Calcium and integrin binding family member 3 (CIB3) variant and a Peptidase mitochondrial processing subunit alpha (PMPCA) variant.

[0009] In certain embodiments, the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene. The change in the nucleotide sequence can, for example, be a substitution, an insertion, a deletion, or a combination thereof. In certain embodiments, the substitution, the insertion, the deletion, or a combination thereof is in a 5’ untranslated region of the gene, an intron of the gene, an exon of the gene, a 3’ untranslated region of the gene, or a combination thereof. The substitution, the insertion, the deletion, or a combination thereof can, for example, be in a regulatory region of the gene.

[0010] In certain embodiments, the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:53-65 and combinations thereof. In certain embodiments, the at least one direwolf gene4908-6027-7382.1 2Attorney Docket No.: 069296.11210 / 28WO2variant associated with coat composition comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122-134, and combinations thereof.

[0011] In certain embodiments, the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:66-67, and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-136, and combinations thereof.

[0012] In certain embodiments, the transgenic animal comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants.

[0013] In certain embodiments, the transgenic animal fails to express an endogenous homologue of at least one of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA. In certain embodiments, the transgenic animal fails to express the endogenous homologue of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0014] In certain embodiments, the transgenic animal is a canine. In certain embodiments, the canine is selected from a short-eared dog (Atelocynus microtis), an African wolf (Cams lupaster), a coyote (Canis latrans), a dog (Canis familiaris), an Ethiopian wolf (Canis simensis), a golden jackal (Canis aureus), a gray wolf (Canis lupus), a crab-eating fox (Cerdocyon thous), a maned wolf (Chrysocyon brachyurus), a dhole (Cuon alpinus), a Falkland island wolf (Dusicyon australis), a South American fox (Dusicyon avus), a black-backed jackal (Lupulella mesomeias), a side-striped jackal (Lupulella adustus), a culpeo (Lycalopex culpeo), a Darwin’s fox (Lycalopex fulvipes), a hoary fox (Lycalopex vetulus), a pampas fox (Lycalopex gymnocercus), a Sechuran fox (Lycalopex sechurae), a South American gray fox (Lycalopex griseus), or an African wild dog (Lycaon pictus).

[0015] Also provided herein are recombinant host cells comprising at least one direwolf (Aenocyon dirus) gene variant associated with (a) coat composition; and / or (b) vibrissae.

[0016] In certain embodiments, the at least one direwolf (Aenocyon dirus) gene variant associated with coat composition is selected from the group consisting of a CBD103 variant, a FGF5 variant, a MC1R variant, a MFSD12 variant, an ASIP variant, a CORIN variant, a DOCK74908-6027-7382.1 3Attorney Docket No.: 069296.11210 / 28WO2variant, a KRT71 variant, a KRT76 variant, a MITF variant, a MLPH variant, a MREG variant, and a TYRP1 variant. In certain embodiments, the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.

[0017] In certain embodiments, the at least one direwolf (Aenocyon dims) gene variant associated with vibrissae is selected from a CIB3 variant and a PMPCA variant.

[0018] In certain embodiments, the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene. The change in the nucleotide sequence can, for example, be a substitution, an insertion, a deletion, or a combination thereof. In certain embodiments, the substitution, the insertion, the deletion, or a combination thereof is in a 5’ untranslated region of the gene, an intron of the gene, an exon of the gene, a 3’ untranslated region of the gene, or a combination thereof. The substitution, the insertion, the deletion, or a combination thereof can, for example, be in a regulatory region of the gene.

[0019] In certain embodiments, the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:53-65 and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122-134, and combinations thereof.

[0020] In certain embodiments, the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:66-67, and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-136, and combinations thereof.

[0021] In certain embodiments, the recombinant host cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants.

[0022] In certain embodiments, the recombinant host cell fails to express an endogenous homologue of at least one of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA. In certain embodiments, the recombinant host cell fails to express the endogenous homologue of at least 2, 3, 4, 5, 6, 7, 8,4908-6027-7382.1 4Attorney Docket No.: 069296.11210 / 28WO29, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0023] In certain embodiments, the recombinant host cell is a canine cell. The canine cell can, for example, be selected from a short-eared dog (Atelocymis microtis) cell, an African wolf (Canis lupaster) cell, a coyote (Canis latrans) cell, a dog Canis familiaris) cell, an Ethiopian wolf (Canis simensis) cell, a golden jackal (Canis aureus) cell, a gray wolf (Canis lupus) cell, a crab-eating fox (Cerdocyon thous) cell, a maned wolf (Chrysocyon brachyurus) cell, a dhole (Cuon alpinus) cell, a Falkland island wolf (Dusicyon australis) cell, a South American fox (Dusicyon avus) cell, a black-backed jackal (Lupulella mesomeias) cell, a side-striped jackal (Lupulella adustus) cell, a culpeo (Lycalopex culpeo) cell, a Darwin’s fox (Lycalopex fulvipes) cell, a hoary fox (Lycalopex vetulus) cell, a pampas fox (Lycalopex gymnocercus) cell, a Sechuran fox (Lycalopex sechurae) cell, a South American gray fox (Lycalopex griseus) cell, or an African wild dog (Lycaon pictus) cell.

[0024] In certain embodiments, the recombinant host cell is a stem cell. The stem cell can, for example, be selected from an induced pluripotent stem cell (iPSC), an embryonic stem (ES) cell, or a mesenchymal stem cell (MSC).

[0025] In certain embodiments, the recombinant host cell is a reprogrammed cell.

[0026] In certain embodiments, the recombinant host cell is a fibroblast cell or a mesenchymal cell. In certain embodiments, the recombinant host cell is an endothelial progenitor cell (EPC) or a pericyte. The recombinant host cell can, for example, be selected from the group consisting of a nerve cell, a cartilage cell, a bone cell, a muscle cell, a fat cell, and an epidermal cell.

[0027] Also provided herein are methods of making transgenic animals comprising at least one direwolf (Aenocyon dirus) gene variant, wherein the direwolf gene variant is associated with (a) the coat composition of a direwolf; and / or (b) the vibrissae of the direwolf.

[0028] In certain embodiments, the methods comprise (a) obtaining a cell from the animal; (b) introducing into the cell at least one direwolf gene variant associated with coat composition and / or at least one direwolf gene variant associated with vibrissae; whereby introducing that at least one direwolf gene variant into the cell produces a recombinant cell; and (c) utilizing the recombinant cell to produce a transgenic animal; wherein the transgenic animal has the coat composition of a direwolf and / or the vibrissae of a direwolf.4908-6027-7382.1 5Attorney Docket No.: 069296.11210 / 28WO2

[0029] In certain embodiments, the at least one direwolf (Aenocyon dints)' gene variant associated with coat composition is selected from the group consisting of a CBD103 variant, a FGF5 variant, a MC1R variant, a MFSD12 variant, an ASIP variant, a CORIN variant, a DOCK7 variant, a KRT71 variant, a KRT76 variant, a MITF variant, a MLPH variant, a MREG variant, and a TYRP1 variant. In certain embodiments, the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.

[0030] In certain embodiments, the at least one direwolf (Aenocyon dints) gene variant associated with vibrissae is selected from a CIB3 variant and a PMPCA variant.

[0031] In certain embodiments, the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene. The change in the nucleotide sequence can, for example, be a substitution, an insertion, a deletion, or a combination thereof. In certain embodiments, the substitution, the insertion, the deletion, or a combination thereof is in a 5’ untranslated region of the gene, an intron of the gene, an exon of the gene, a 3’ untranslated region of the gene, or a combination thereof. The substitution, the insertion, the deletion, or a combination thereof can, for example, be in a regulatory region of the gene.

[0032] In certain embodiments, the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:53-65 and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122-134, and combinations thereof.

[0033] In certain embodiments, the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:66-67, and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-136, and combinations thereof.

[0034] In certain embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants are introduced into the cell.

[0035] In certain embodiments, the recombinant cell fails to express an endogenous homologue of at least one of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76,4908-6027-7382.1 6Attorney Docket No.: 069296.11210 / 28WO2MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA. Tn certain embodiments, the recombinant cell fails to express the endogenous homologue of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0036] In certain embodiments, the recombinant cell is a stem cell. The stem cell can, for example, be selected from an induced pluripotent stem cell (iPSC), an embryonic stem (ES) cell, or a mesenchymal stem cell (MSC).

[0037] In certain embodiments, the recombinant cell is a reprogrammed cell.

[0038] In certain embodiments, the recombinant cell is a fibroblast cell or a mesenchymal cell. In certain embodiments, the recombinant cell is an endothelial progenitor cell (EPC) or a pericyte. The recombinant cell can, for example, be selected from the group consisting of a nerve cell, a cartilage cell, a bone cell, a muscle cell, a fat cell, and an epidermal cell.

[0039] In certain embodiments, the animal is a canine. The canine can, for example, be selected from a short-eared dog (Atelocynus microtis), an African wolf Canis lupaster), a coyote (Cams latrans), a dog (Canis familiaris), an Ethiopian wolf (Canis simensis), a golden jackal Canis aureus), a gray wolf Canis lupus), a crab-eating fox (Cerdocyon thous), a maned wolf (Chrysocyon brachyurus), a dhole (Cuon alpinus), a Falkland island wolf (Dusicyon australis), a South American fox Dusicyon avus), a black-backed jackal (Lupulella mesomeias), a side- striped jackal (Lupulella adustus), a culpeo (Lycalopex culpeo), a Darwin’s fox (Lycalopex fulvipes), a hoary fox (Lycalopex vetulus), a pampas fox (Lycalopex gymnocercus), a Sechuran fox (Lycalopex sechurae), a South American gray fox (Lycalopex griseus), or an African wild dog (Lycaon pictus).

[0040] Also provided are transgenic animals made by the methods disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, the figures demonstrate embodiments of the present invention. It should be understood, however, that the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

[0042] FIG. 1 shows a schematic of a canid phylogenetic tree. An IQ-Tree (Minh et al., Mol. Biol. Evol. 37:1530-1534 (2020)) method was used to infer locus trees from windows of aligned4908-6027-7382.1 7Attorney Docket No.: 069296.11210 / 28WO2sequences from representative canid species; an ASTRAL (Zhang et al , BMC Bioinformatics 19: 153 (2018)) method was used to infer consensus tree of canid species, and a FigTree (Ramabaut, FigTree v 1.3.1, Institute of Evolutionary Biology, University of Edinburgh; tree.bio.ed.ac.uk / software / figtree / (2010)) method was used for generating plot.

[0043] FIG. 2 shows a schematic demonstrating canid genome sequence distance. A mash (Ondov et al., Genome Biol. 17:132 (2016)) method was used for sequence distance estimation from k-mers; BWA (Li and Durbin, Bioinformatics 25:1754-1760 (2009)) methods were used for aligning sequencing reads; GATK (Auwera and O’Connor, Genomics in the cloud: using Docker, GATK, and WDL in Terra. First edition. Beijing Boston Farnham Sebastopol Tokyo: O’Reilly (2020)) methods were used for calling sequence variants; bcftools (Danecek et al., Gigascience 10:giab008 (2021)) methods were used for variant processing to assess sequence similarity; R tidyverse (Wickham et al., J. Open Source Softw 4: 1686 (2019)) methods were used for generating plot.

[0044] FIG. 3 shows a graph demonstrating enhanced recovery of ancient DNA via the Direwolf (DW) specimens from Gigantobison Bay (DireGB) and Sheriden Pit (DireSP). BWA (Li and Durbin Bioinformatics 25:1754-1760 (2009)) methods were used for aligning sequencing reads; a pilon (Walker et al., PLos ONE 9:el 12963 (2014)) method was used for polishing genome assembly; bcftools (Danecek et al., Gigascience 10:giab008 (2021)) methods were used for calling pseudohaploid consensus sequence; a mosdepth (Pedersen and Quinlan, Bioinformatics 34:867-868 (2018)) method was used for assessing depth and breadth of sequencing reads mapped to reconstructed sequence; and R tidyverse (Wickham et al., J. Open Source Softw 4: 1686 (2019)) methods were used for generating plot.

[0045] FIG. 4 shows the amino acid and 3D structural comparison between proteins encoded by two genes of interest, MITF and LCORL, in both gray wolf and direwolf. DeepFRI (Gligorijevic et al., Nat. Commun. 12:3168 (2021)) and AlphaFold2 (Jumper et al., Nature 596:583-589 (2021)) methods were used for predicting protein functional domains and structure models and generating plots; US-align (Zhang et al., Nat. Methods 19: 1109-1115 (2022)) methods were used for comparing protein structures; and Mol* Viewer (Sehnal et al., Nucleic Acids Res. 49:W431-W437 (2021)) was used for generating plot.

[0046] FIG. 5 shows images of adherent endothelial progenitor cells (EPCs) (left) and pericytes (right) derived from peripheral blood of adult gray wolves (Canis lupus).4908-6027-7382.1 8Attorney Docket No.: 069296.11210 / 28WO2

[0047] FIG. 6 shows an image of cleavage of genetically engineered gray wolf embryos produced by interspecies somatic cell nuclear transfer (iSCNT) with EPCs.

[0048] FIG. 7 shows an image of an ultrasound providing confirmation of a successful pregnancy with a transgenic wolf fetus.

[0049] FIG. 8 shows an image of a heatmap of 50 of the most variable genes between unedited and genetically edited wolf pups in the context of publicly available series of RNA sequencing from dog pups. The TrimGalore (Krueger et al., FelixKrueger / TrimGalore: v0.6.10 zenodo.org / record / 5127898 (2023)) method was used to trim mRNA sequencing reads; STAR (Dobin et al., Bioinformatics 29:15-21 (2013)) method was used to align mRNA sequencing reads to wolf reference genome assembly; FeatureCount (Liao et al., Bioinformatics 30:923-930 (2014)) method was used to generate raw gene counts; StringTie (Kovaka et al., Genome Biol.20:278 (2019)) method was used to normalize for expression analysis of mRNA sequencing data; EdgeR (Robinson et al., Bioinformatics 26:139-140 (2010)), DESeq2 (Love et al., Genome Biol. 15:550 (2014)), and Isoform SwitchAnalyzeR (Vitting-Seerup and Sandelin, Bioinformatics 35:4469-4471 (2019)) methods were used for detecting differentially expressed genes and transcripts; and the R package clusterProfder (Yu et al., J. Integr. Biol. 16:284-287 (2012)) method was used for clustering expression profdes and generating plot. Publicly available dog fetal RNA sequencing data was from BarkBase (Megquier et al. 2019).

[0050] FIGs. 9A-9F show photographs of the development of the gene-edited direwolves. FIG.9A shows a photograph of the direwolf pups at 15 days. FIG. 9B shows a photograph of a direwolf pup at 1 month. FIG. 9C shows a photograph of a direwolf pup at 2 months. FIGs. 9D and 9E show photographs of direwolves at 3 months. FIG. 9F shows a photograph of the direwolves at 5 months.DETAILED DESCRIPTION OF THE INVENTION

[0051] This disclosure is based on the discovery that using only a minimal number of genetic enhancers it is possible to generate a transgenic animal with the coat composition and / or vibrissae of a direwolf. Accordingly, the disclosure provides methods of generating transgenic animals by introducing at least one direwolf gene variant associated with coat composition and / or vibrissae. The at least one direwolf gene variant associated with coat composition can, for example, be a CBD103 variant, a FGF5 variant, a MC1R variant, a MFSD12 variant, an ASIP variant, a CORIN variant, a DOCK7 variant, a KRT71 variant, a KRT76 variant, a MITF4908-6027-7382.1 9Attorney Docket No.: 069296.11210 / 28WO2variant, a MLPH variant, a MREG variant, and a TYRP1 variant. The at least one direwolf gene variant associated with vibrissae is selected from a CIB3 variant and a PMPCA variant.

[0052] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0053] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments, or applications of the present invention.Definitions

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification.

[0055] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0056] Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0057] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.4908-6027-7382.1 10Attorney Docket No.: 069296.11210 / 28WO2

[0058] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0059] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and / or.”

[0060] As used herein, the term “consists of,” or variations such as “consist of’ or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, but that no additional integer or group of integers can be added to the specified method, structure, or composition.

[0061] As used herein, the term “consists essentially of,” or variations such as “consist essentially of’ or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure, or composition. See M.P.E.P. § 2111.03.

[0062] The words “right,” “left,” “lower,” and “upper” designate directions in the drawings to which reference is made.4908-6027-7382.1 11Attorney Docket No.: 069296.11210 / 28WO2

[0063] It should also be understood that the terms “about,” “approximately,” “generally,” “substantially” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

[0064] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0065] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0066] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. AppL Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat’L Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0067] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-4908-6027-7382.1 12Attorney Docket No.: 069296.11210 / 28WO23402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0068] Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0069] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0070] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a4908-6027-7382.1 13Attorney Docket No.: 069296.11210 / 28WO2polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0071] As used herein, the term “polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0072] As used herein, the term “vector” is a replicon in which another nucleic acid segment can be operably inserted so as to bring about the replication or expression of the segment.

[0073] As used herein, the term “host cell” refers to a cell comprising a nucleic acid molecule of the present disclosure, such as, for example an isolated vector comprising an isolated nucleic acid of the invention. The “host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a “host cell” is a cell transfected with a nucleic acid molecule of the invention to become, e.g., a recombinant host cell. In another embodiment, a “host cell” is a progeny or potential progeny of such a transfected cell. A progeny of a cell may or may not be identical to the parent cell, e.g., due to mutations or environmental influences that can occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome. A host cell can be, for example, any type of4908-6027-7382.1 14Attorney Docket No.: 069296.11210 / 28WO2prokaryotic, eukaryotic, or archaeal cell. In some instances, the host cell is a bacterial cell. Tn some instances, the host cell is a mammalian cell. In some embodiments, the mammalian host cell is a canine cell.

[0074] The term “expression” as used herein, refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications.

[0075] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms “peptide,” “polypeptide,” and “protein” can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0076] The peptide sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of the amino acids are known, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.

[0077] The term “heterologous nucleic acid” or “heterologous polypeptide” refers to a nucleic acid or a polypeptide whose sequence is not identical to that of another nucleic acid or polypeptide naturally found in the same host cell or the same host. As use herein, the “heterologous nucleic acid” or “heterologous polypeptide” can be heterologous to the bacterial cell and / or the mammalian host.

[0078] As used herein, the term “transform” or “transformation” refers to the transfer of a nucleic acid fragment into a host cell, such as a host bacterial cell, resulting in genetically-stable4908-6027-7382.1 15Attorney Docket No.: 069296.11210 / 28WO2inheritance. Host cells comprising the transformed nucleic acid fragment are referred to as “recombinant” or “transgenic” or “transformed” organisms.

[0079] As used herein, the term “isolated” means a biological component (such as a nucleic acid, peptide, or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. “Isolated” nucleic acids, peptides and proteins can be part of a composition and still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0080] As used herein, “gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.

[0081] As used herein, a “promoter” is an example of a transcriptional regulatory sequence and is specifically a nucleic acid sequence generally described as the proximal region of a gene located 5' to the start codon. The transcription of an adjacent nucleic acid segment is initiated at the promoter region. A repressible promoter's rate of transcription decreases in response to a repressing agent. An inducible promoter's rate of transcription increases in response to an inducing agent. A constitutive promoter's rate of transcription is not specifically regulated, though it can vary under the influence of general metabolic conditions.

[0082] The term “gene product,” as used herein, refers to any product encoded by a nucleic acid sequence. Accordingly, a gene product may, for example, be a primary transcript, a mature transcript, a processed transcript, or a protein or peptide encoded by a transcript. Examples for gene products, accordingly, include mRNAs, rRNAs, hairpin RNAs (e.g., microRNAs, shRNAs, siRNAs, tRNAs), and peptides and proteins, for example, reporter proteins or therapeutic proteins.

[0083] As used herein, the phrase “coat composition” refers to the nature and quality of the fur of the transgenic animal comprising at least one direwolf gene variant. The coat composition can comprise different types of hair, fur, or wool, and the coat composition can have different textures ranging from downy and smooth to spiky. Additionally, the coat composition can have4908-6027-7382.1 16Attorney Docket No.: 069296.11210 / 28WO2multiple coats, which can include an undercoat and a topcoat. As disclosed herein, the coat composition of the transgenic animal comprising at least one direwolf gene variant would resemble the coat composition of a direwolf.

[0084] As used herein, the phrase “vibrissae” refers to whiskers of the transgenic animal comprising the at least one direwolf gene variant. The whiskers are specialized hairs that help the transgenic animal sense the environment. The vibrissae are a source of tactile sensory information for the transgenic animal. As used herein the vibrissae of the transgenic animal comprising at least one direwolf gene variant would resemble the vibrissae of a direwolf.

[0085] As used herein, the term “stem cell” refers to a cell that can self-renew and differentiate to at least one more-differentiated or less developmentally-capable phenotype. The term “stem cell” encompasses stem cell lines, induced stem cells, non-human embryonic stem cells, pluripotent stem cells, multipotent stem cells, amniotic stem cells, placental stem cells, or adult stem cells. An “induced stem cell” is one derived from a non-pluripotent cell induced to a less-differentiated or more developmentally-capable phenotype by introduction of one or more reprogramming factors or genes. As the term is used herein, an induced stem cell need not be pluripotent, but has the capacity to differentiate, under appropriate conditions, to more than one more-highly-differentiated phenotype. It should be understood that the capacity was not present prior to the introduction of reprogramming factors. An induced stem cell will express at least one stem cell marker not expressed by the parent cell prior to introduction of reprogramming factors. In this context, a stem cell marker is exclusive of a factor introduced by reprogramming. An induced pluripotent stem cell, or iPS cell, has the induced capacity to differentiate, under appropriate conditions, to a cell phenotype derived from each of the endoderm, mesoderm, and ectoderm germ layers.

[0086] The term “marker” as used herein is used to describe a characteristic and / or phenotype of a cell. Markers can be used for selection of cells comprising characteristics of interest and can vary with specific cells. Markers are characteristics, whether morphological, structural, functional, or biochemical (enzymatic) characteristics of the cell of a particular cell type, or molecules expressed by the cell type. In one aspect, such markers are proteins. Such proteins can possess an epitope for antibodies or other binding molecules available in the art. However, a marker can consist of any molecule found in or on a cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of4908-6027-7382.1 17Attorney Docket No.: 069296.11210 / 28WO2morphological characteristics or traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, and the ability to differentiate along particular lineages. Markers can be detected by any method available to one of skill in the art. Markers can also be the absence of a morphological characteristic or absence of proteins, lipids etc. Markers can be a combination of a panel of unique characteristics of the presence and / or absence of polypeptides and other morphological or structural characteristics. In one embodiment, the marker is a cell surface marker.

[0087] The term “exogenous” refers to a substance present in a cell that was introduced by the hand of man. The term “exogenous” when used herein can refer to a nucleic acid (e. ., a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively lower amounts and in which one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels.

[0088] As used herein, the term “reprogramming genes” or “reprogramming factors” refers to agents or nucleic acid molecules that can induce the reprogramming process in a somatic cell to re-express a less-differentiated, more stem-cell like phenotype. The reprogramming factor can be a nucleic acid, a polypeptide, or a small molecule that promotes a reprogrammed phenotype when introduced to a cell. Non-limiting examples of reprogramming factors include: Oct4 (Octamer binding transcription factor-4), SOX2 (Sex determining region Y)-box 2, Klf4 (Kruppel Like Factor-4), and c-Myc. These are the so-called “classical” or “standard” set of reprogramming factors used to derive, for example, induced pluripotent stem cells. Additional factors that can be considered reprogramming factors when introduced in the process of reprogramming cells to a less differentiated or stem cell phenotype include LIN28 + Nanog, Esrrb, Pax5 shRNA, C / EBPa, p53 siRNA, UTF1, DNMT shRNA, Wnt3a, SV40 LT(T), hTERT, small molecule chemical agents including, but not limited to BIX-01294, BayK8644, RG108,4908-6027-7382.1 18Attorney Docket No.: 069296.11210 / 28WO2, dexamethasone, VP A, TSA, SAHA, PD0325901 + CHIR99021(2i) and A-83-01. Tn some embodiments, the reprogramming genes or factors are Oct4, Klf4, SOX2, and c-Myc.

[0089] As used herein, the terms “dedifferentiation” or “retrodifferentiation” or “reprogramming” refer to a process that generates a cell that re-expresses a less differentiated phenotype than the cell from which it is derived and / or expresses at least one stem cell marker not expressed prior to that process. For example, a terminally-differentiated cell can be dedifferentiated to a multipotent cell. That is, dedifferentiation shifts a cell backward along the differentiation spectrum of totipotent cells to fully differentiated cells. Typically, reversal of the differentiation phenotype of a cell requires artificial manipulation of the cell, for example, by introducing or expressing exogenous polypeptide factors. Reprogramming is not typically observed under native conditions in vivo or in vitro.

[0090] As used herein, a “reprogrammed cell” is a cell that has been contacted with one or more reprogramming factors and expresses a less differentiated phenotype than the cell from which it was derived. The reprogrammed cell can also have the capacity to self-renew and will express at least one stem cell marker that was not delivered to the cell as a reprogramming factor.Furthermore, the reprogrammed cell will have the capacity to differentiate into a more-differentiated somatic cell type following differentiation protocols provided herein or described in the art.

[0091] As used herein, the term “somatic cell” refers to any cell other than a germ cell, a cell present in or obtained from a pre-implantation embryo, or a cell resulting from proliferation of such a cell in vitro. Stated another way, a somatic cell refers to any cells forming the body of an organism, excluding germ cells. Every cell type in the mammalian body-apart from the sperm and ova and the cells from which they are made (gametocytes)-is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all substantially made up of somatic cells. In some embodiments the somatic cell is a “non-embryonic somatic cell,” by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from proliferation of such a cell in vitro. In some embodiments the somatic cell is an “adult somatic cell,” by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro.Nucleic Acids, Vectors, Recombinant Cells, and Transgenic Animals Expressing Direwolf Specific Gene Variants4908-6027-7382.1 19Attorney Docket No.: 069296.11210 / 28WO2

[0092] The generation of direwolves and the restoration of a population of this extinct species can have a wide array of benefits, including, but not limited to, conservation and de-extinction efforts, as the methods used herein can restore an extinct species and preserve species diversity to combat climate change effects. These methods can also be used to preserve existing species with coat composition similar to direwolves and / or vibrissae similar to direwolves and likely confer beneficial adaptations to habitats, as well as preserve existing environments through rewilding efforts. Finally, as part of the genomic editing methods described herein, novel genomic information about living canid species as well as the extinct direwolf has been produced. This information can aid in the research efforts around evolutionary biology, speciation, and ecology. Finally, the de-extinction of direwolf will advance synthetic biology and will serve as a precedent for advanced multiplexed editing in organisms.

[0093] The isolated nucleic acids, vectors, recombinant cells, and transgenic animals described herein are based, in part, on the discovery that cells (e.g., a short-eared dog (Atelocynus microtis) cell, an African wolf (Canis lupaster) cell, a coyote (Canis latrans) cell, a dog (Canis familiaris) cell, an Ethiopian wolf (Canis simensis) cell, a golden jackal (Canis aureus) cell, a gray wolf (Canis lupus) cell, a crab-eating fox (Cerdocyon thous) cell, a maned wolf (Chrysocyon brachyurus) cell, a dhole (Cuon alpinus) cell, a Falkland island wolf (Dusicyon australis) cell, a South American fox (Dusicyon avus) cell, a black-backed jackal (Lupulella mesomeias) cell, a side-striped jackal (Lupulella adustus) cell, a culpeo (Lycalopex culpeo) cell, a Darwin’s fox (Lycalopex fulvipes) cell, a hoary fox (Lycalopex vetulus) cell, a pampas fox (Lycalopex gymnocercus) cell, a Sechuran fox (Lycalopex sechurae) cell, a South American gray fox (Lycalopex griseus) cell, or an African wild dog (Lycaon pictus) cell) can be modified to comprise and express alleles or homologues from the direwolf e.g., Aenocyon dims). In particular, viable cells can be gene-edited, whether by transfection, transduction, or modification of existing canine (e.g., wolf) homologues to mimic the phenotypes of the direwolf gene variants or alleles of the direwolf genes. In some embodiments, the endogenous homologues of the direwolf genes are deleted or inactivated. Similar modifications to introduce direwolf genes can be made to viable cells of other, non-human relatives of the canines (e.g., wolves). The direwolf variants or alleles can modify the phenotype of the gene edited cells. The isolated nucleic acids, vectors, recombinant cells, and transgenic animals described herein provide a synthetic4908-6027-7382.1 20Attorney Docket No.: 069296.11210 / 28WO2alternative to wildlife products and new tools for understanding genetic diversity and cellular biology in endangered and extinct species of wildlife.

[0094] In one aspect, described herein is at least one exogenous nucleic acid sequence encoding a direwolf gene, or comprising a modification of an endogenous gene to express a direwolf homologue or variant of the endogenous gene. Of particular interest are genes that are shared by every direwolf genome sequenced, which are not shared by any canine (e.g., wolf) genome sequenced. By choosing genes in this manner, effects of individual variation within the group of direwolf genomes sequenced and variations in canine genomes are minimized to focus on those variant sequences that are fully direwolf. In view of this, as used herein, a “direwolf gene,” “direwolf gene variant” or “direwolf homologue” is a gene encoding a polypeptide that has a sequence encoded by all direwolf genomes sequenced, and which differs from the homologous polypeptide encoded in all canine genomes sequenced. In this context, “differs from” refers to a difference of at least one amino acid relative to the homologous polypeptides encoded by the canine. A non-coding or regulatory nucleic acid sequence can be considered a “direwolf sequence” if a non-coding motif of at least 20 nucleotides is present in every direwolf genome sequenced, and not present in any canine genome sequenced, or if one or more nucleotides in the regulatory region of every direwolf genome sequence differs from one or more nucleotides in the regulatory region of the canine genome. A canine gene or sequence modified by human intervention to encode a direwolf gene or gene variant sequence is a direwolf gene or gene variant as the term is used herein. Where a direwolf gene or gene variant as referred to herein is only found encoded in a direwolf genome, and where the direwolf is extinct, a direwolf gene or gene variant sequence is necessarily exogenous to a viable cell; that is, the direwolf gene or gene variant sequence is “exogenous” whether the sequence is in the cell through introduction of a foreign sequence or through gene editing an endogenous sequence to encode the direwolf gene or gene variant sequence.

[0095] As identified herein, the direwolf variants in the application were aligned to the canine reference (28x PacBio data, 10X genomics chromium data, and dovetail Hi-C data generated by Darwin Tree of Life Project (www.darwintreeoflife.org)). The relevant gene sequences of the canine reference are provided herein. As indicated in Table 1, target genes and loci (“Gene”) harboring the DNA sequence variation associated with species phenotypes of interest from Canis lupus. Canis lupus familiar is., and Aenocyon dims, where Canis lupus is used as the reference4908-6027-7382.1 21Attorney Docket No.: 069296.11210 / 28WO2genome. Reported for each gene is the associated phenotype (“Phenotype”) supporting selection for genome editing (Webster et al. 2015; Plassais et al. 2017; Shu et al. 2017; Claes et al. 2018; Schweizer et al. 2018; Plassais et al. 2019; Huang et al. 2021; Graber et al. 2022; Wagner et al.2022; Majeres et al. 2024; Rajderkar et al. 2024). Shown are the SEQ ID NOs for representative sequences of the extant gray wolf genome (“Canine SEQ ID NO”) and the phenotype-edited DNA sequences (“Direwolf SEQ ID NO”) of the extinct dire wolf genome to which template sequences can be edited towards at target genomic loci. For the template DNA sequences, representative DNA sequences for 69 target loci (SEQ ID NOs: 1-69) from the gray wolf reference genome (NCBI GenBank accession GCA 905319855.2) including sequence variation present in the template genome of the donor animal represented by IUPAC nucleotide codes (see “IUPAC Nucleotide Code”). For the phenotype-directed editing, there are modified DNA sequences for 7 target loci (SEQ ID NOs:70-72 and 122-125). For sequence de-extinction, there are reconstructed DNA sequences for 62 target loci (SEQ ID NOs:73-121 and 126-138) from ancient dire wolf genome progressively edited from gray wolf template sequence excluding sequence bases unsupported by ancient DNA evidence as represented by IUPAC nucleotide codes (see, e.g., “IUPAC Nucleotide Code”). The table lists the number of species-specific DNA sequence variants selected for editing within protein-coding regions and regulatory regions of gene sequences, including 5’ untranslated regions, 3’ untranslated regions, intronic sequences, and putative canine regulatory elements such as promoters, enhancers, or repressors (“Total variants in gene”).

[0096] As used herein, the phrase “IUPAC Nucleotide Code” refers to the IUPAC Nucleotide Code representing both determinate (“A” for adenosine; “T” for thymine; “C” for cytosine; “G” for guanine) and indeterminate (“N” for any base; “K” for guanine or thymine; “R” for adenosine or guanine; “Y” for cytosine or thymine; “S” for guanine or cytosine; “W” for adenosine or thymine; “M” for adenosine or cytosine; “B” for cytosine, guanine, or thymine; “D” for adenosine, guanine, or thymine; “H” for adenosine, cytosine, or thymine; “V” for adenosine, cytosine, or guanine) nucleotide bases. For template DNA sequences from the donor gray wolf genome, indeterminate nucleotide codes reflect an individual’s maternal and paternal genetic variation from reference gray wolf genome assembly. For reconstructed DNA sequences from the ancient genome of the extinct dire wolf, indeterminate nucleotide “N” represents DNA4908-6027-7382.1 22Attorney Docket No.: 069296.11210 / 28WO2sequences not well-evidenced from ancient DNA sequencing data, which will be left as extant gray wolf sequence,

[0097] Thus, provided herein are isolated nucleic acid sequences comprising at least one direwolf (Aenocyon dints) gene variant associated with (a) coat composition; and / or (b) vibrissae. The isolated nucleic acid sequences can, for example, comprise at least one direwolf gene variant associated with coat composition selected from the group consisting of a CBD103 variant, a FGF5 variant, a MC1R variant, a MFSD12 variant, an ASIP variant, a CORIN variant, a DOCK7 variant, a KRT71 variant, a KRT76 variant, a MITF variant, a MLPH variant, a MREG variant, and a TYRP1 variant. In certain embodiments, the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.

[0098] Also provided are isolated nucleic acid sequences comprising at least one direwolf (Aenocyon dirus) gene variant associated with vibrissae selected from a CIB3 variant and a PMPCA variant.

[0099] In certain embodiments, the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene. The change in the nucleotide sequence can, for example, be a substitution, an insertion, a deletion, or a combination thereof. The substitution, the insertion, the deletion, or a combination thereof can, for example, be in a 5’ untranslated region of the gene, an intron of the gene, an exon of the gene, a 3’ untranslated region of the gene, or a combination thereof. The substitution, the insertion, the deletion, or a combination thereof can, for example, be in a regulatory region of the gene.

[0100] In certain embodiments, a direwolf gene variant can comprise one or more substitutions within the regulatory region and / or within the coding region of the gene as compared to the corresponding reference genome. Thus, the direwolf gene variant can comprise at least 1, at least 5, at least 10, at least 20, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, or at least 3000 substitutions within the regulatory region and / or within the coding region of the gene. The direwolf gene variant can comprise about 1 to about 3000, about 25 to about 2750, about 50 to about 2500, about 75 to about 2250, about 100 to about 2000, about 150 to about 1750, about 200 to about 1500, about 300 to about 1250, or about 400 to4908-6027-7382.1 23Attorney Docket No.: 069296.11210 / 28WO2about 1000, about 500 to about 750 substitutions, and any value in between in the regulatory region and / or within the coding region of the gene.

[0101] In certain embodiments, the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:53-65, and combinations thereof. The at least one direwolf gene variant associated with coat composition can, for example, comprise a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:53-65, and combinations thereof. The at least one direwolf gene variant associated with coat composition can, for example, comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122-134, and combinations thereof.

[0102] In certain embodiments, the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:66-67 and combinations thereof. The at least one direwolf gene variant associated with vibrissae can, for example, comprise a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 66-67, and combinations thereof. The at least one direwolf gene variant associated with vibrissae can, for example, comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-136, and combinations thereof.

[0103] In certain embodiments, the isolated nucleic acids comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants.

[0104] The exact location and substitution of a direwolf gene variant can, for example, be determined by performing an alignment with the direwolf sequence and canine sequence provided herein in Table 1. The direwolf gene variant can comprise at least one substitution, or more than one substitution, as compared to the canine (e.g., wolf) sequence. Thus, by way of an example, a direwolf gene variant for ASIP can, for example, comprise 1, 5, 10, 15, 20, 30, 40, 45, 50, 55, 56 (or any number in between) substitutions in the gene, including in the intron of the gene, the exon of the gene, and / or the upstream / downstream regulatory regions of the gene when4908-6027-7382.1 24Attorney Docket No.: 069296.11210 / 28WO2compared to the canine sequence. By way of another example, a direwolf gene variant for CIB3 can, for example, comprise 1, 5, 10, 15, 20, 30, 31, 32, 33, 34, 35, 36 (or any number in between) substitutions in the gene, including in the intron of the gene, the exon of the gene, and / or the upstream / downstream regulatory regions of the gene when compared to the canine sequence. A person skilled in the art will understand how to identify the substitutions and total number of substitutions using the direwolf gene variant sequences and canine sequences provided in Table 1.4908-6027-7382.1 25Atorney Docket No.: 069296.11210 / 28WO24908-6027-7382.1 26Attorney Docket No.: 069296.11210 / 28WO2Total Variants in Gene can include variants in introns, exons, and regulatory regions.

[0105] Also provided are isolated vectors comprising the isolated nucleic acid sequences described herein.

[0106] Also provided are recombinant host cells comprising at least one direwolf (Aenocyon dims) gene variant associated with (a) coat composition; and / or (b) vibrissae.

[0107] In certain embodiments, the at least one direwolf (Aenocyon dims) gene variant associated with coat composition is selected from the group consisting of a CBD103 variant, a FGF5 variant, a MC1R variant, a MFSD12 variant, an ASIP variant, a CORIN variant, a DOCK7 variant, a KRT71 variant, a KRT76 variant, a MITF variant, a MLPH variant, a MREG variant, and a TYRP1 variant. In certain embodiments, the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.

[0108] In certain embodiments, the at least one direwolf (Aenocyon dims) gene variant associated with vibrissae is selected from a CIB3 variant and a PMPCA variant.

[0109] In certain embodiments, the recombinant host cell comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants. In some embodiments, the direwolf gene variant comprises one or more substitutions within the regulatory region and / or within the coding region of the gene as compared to a corresponding reference genome, wherein the reference genome is the genome of the host cell prior to the one or more substitutions used to generate the recombinant cell.4908-6027-7382.1 27Attorney Docket No.: 069296.11210 / 28WO2

[0110] In certain embodiments, the recombinant host cell fails to express an endogenous homologue of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA. In certain embodiments, the recombinant host cell fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0111] Also provided are recombinant host cells comprising at least one of the isolated nucleic acid sequences described herein. In certain embodiments, the recombinant host cell comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the isolated nucleic acids described herein.

[0112] In certain embodiments, the recombinant host cell is a canine cell. In certain embodiments, the recombinant host cell is a short-eared dog (Atelocynus microtis) cell, an African wolf (Canis lupaster) cell, a coyote (Canis latrans) cell, a dog (Canis familiaris) cell, an Ethiopian wolf (Canis simensis) cell, a golden jackal (Canis aureus) cell, a gray wolf (Canis lupus) cell, a crab-eating fox (Cerdocyon thous) cell, a maned wolf (Chrysocyon brachyurus) cell, a dhole (Cuon alpinus) cell, a Falkland island wolf (Dusicyon australis) cell, a South American fox (Dusicyon avus) cell, a black-backed jackal (Lupulella mesomeias) cell, a side-striped j ackal (Lupulella adustus) cell, a culpeo (Lycalopex culpeo) cell, a Darwin’s fox (Lycalopex fulvipes) cell, a hoary fox (Lycalopex vetulus) cell, a pampas fox (Lycalopex gymnocercus) cell, a Sechuran fox (Lycalopex sechurae) cell, a South American gray fox (Lycalopex griseus) cell, or an African wild dog (Lycaon pictus) cell.

[0113] The direwolf gene variants described herein can be used in any combination to be expressed in any recombinant host cell as described herein.Cells

[0114] The direwolf gene variants described herein can be expressed by any viable cell that can accept exogenous genetic material. The cell can be, for example, a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is a eukaryotic cell. The cell can be a reprogrammed cell, a non-human oocyte, a cell of a non-human embryo or a cell of a non-human blastula. In some embodiments of any of the aspects, the cell is a fibroblast cell. In some embodiments, the cell is selected from the group consisting of a nerve cell, a cartilage cell, a bone cell, a muscle cell, a bone cell, a fat cell, and an epidermal cell. In some embodiments, the4908-6027-7382.1 28Attorney Docket No.: 069296.11210 / 28WO2cell was previously differentiated into a cell selected from the group consisting of a nerve cell, cartilage cell, bone cell, muscle cell, bone cell, fat cell, and an epidermal cell.

[0115] The scientific literature provides guidance for one of ordinary skill in the art to isolate and prepare cells as necessary for use with the isolated nucleic acids and vectors described herein.

[0116] The cells described herein can be from any viable non-human source or organism.Usually, the organism is an animal or vertebrate such as a wild animal, zoo animal, endangered animal, rodent, domestic animal, or bird. Animals can include, as non-limiting examples, a dunnart, hippopotamus, hyrax, manatee, bear, panda, feline species, e.g., tiger, lion, cheetah, bobcat, canine species, e.g., fox, wolf, avian species, e.g., ostrich, emu, penguin, pigeon, and fish, e.g., trout, catfish, and salmon. In some embodiments, the cell described herein is from a mammal. A non-limiting example of an organism from which cells can be derived includes canines (e.g., a short-eared dog (Atelocynus microtis), an African wolf (Canis lupastef), a coyote (Cams latrans), a dog (Canis familiaris), an Ethiopian wolf (Canis simensis), a golden jackal (Canis aureus), a gray wolf (Canis lupus), a crab-eating fox (Cerdocyon thous), a maned wolf (Chrysocyon brachyurus), a dhole (Cuon alpinus), a Falkland island wolf (Dusicyon australis), a South American fox (Dusicyon avus), a black-backed jackal (Lupulella mesomeias), a side- striped jackal (Lupulella adustus), a culpeo (Lycalopex culpeo), a Darwin’s fox (Lycalopex fulvipes) , a hoary fox (Lycalopex vetulus), a pampas fox (Lycalopex gymnocercus), a Sechuran fox (Lycalopex sechurae), a South American gray fox (Lycalopex griseus), or an African wild dog (Lycaon pictus) ).

[0117] In certain embodiments, a cell useful in the methods and compositions described herein is a canine cell. In some embodiments, the cell is a canine fibroblast cell. In some embodiments, the cell is a canine stem cell. In some embodiments, the cell described herein is a canine somatic cell reprogrammed to a stem cell or stem cell-like phenotype having stem cell-like morphology and / or expressing at least one stem cell marker described herein.

[0118] The cells described herein can be from any tissue isolated from an organism by methods known in the art. For example, placental tissue can be isolated from a given organism (e.g., a canine), after full term delivery of young, and subsequently processed for cellular isolation and / or culture by methods known in the art. Additional exemplary cell types that can be used for the compositions and methods described herein include but are not limited to fibroblasts, skin4908-6027-7382.1 29Attorney Docket No.: 069296.11210 / 28WO2cells, blood cells (e.g., leukocytes, monocytes, dendritic cells), stem cells, hematopoietic cells, liver cells, vascular cells, muscle cells, pancreatic cells, neural cells, ocular or retinal cells, epithelial or endothelial cells, lung cells, cardiac cells, intestinal cells, diaphragmatic cells, renal (i.e., kidney) cells, bone marrow cells, or any one or more selected tissues or cells of an organism for which genetic modification or gene editing to express a genetic enhancer and / or a direwolf gene is contemplated.

[0119] In certain embodiments, the isolated nucleic acids and vectors described herein are used in stem cells. Stem cells are cells that retain the ability to renew themselves through mitotic cell division and can differentiate into more specialized cell types. Three broad types of mammalian stem cells include: embryonic stem (ES) cells that are found in blastocysts, induced pluripotent stem cells (iPSCs) that are reprogrammed from somatic cells, and adult stem cells that are found in adult tissues. Other sources of stem cells can include, for example, amnion-derived or placental -derived stem cells. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

[0120] In certain embodiments, the recombinant host cell is a stem cell. The stem cell can, for example, be selected from an induced stem cell, embryonic stem (ES) cell, or a mesenchymal stem cell (MSC). In certain embodiments, the recombinant host cell is a reprogrammed cell. In certain embodiments, the recombinant host cell is a fibroblast cell or a mesenchymal cell. In certain embodiments, the recombinant host cell is an endothelial progenitor cell (EPC) or a pericyte. In certain embodiments, the recombinant host cell is selected from the group consisting of a nerve cell, a cartilage cell, a bone cell, a muscle cell, a bone cell, a fat cell, and an epidermal cell.

[0121] In certain embodiments, the recombinant host cell fails to express an endogenous homologue of at least one direwolf gene variant. In certain embodiments, the recombinant host cell fails to express the endogenous homologue of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA. In certain embodiments, the recombinant host cell fails to express the endogenous homologue of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0122] In certain embodiments, the recombinant host cell is a canine cell. In certain embodiments, the recombinant host cell is a short-eared dog Atelocynus microtis) cell, an4908-6027-7382.1 30Attorney Docket No.: 069296.11210 / 28WO2African wolf (Canis hipasler) cell, a coyote (Canis latrans) cell, a dog (Canis familiaris) cell, an Ethiopian wolf (Canis simensis) cell, a golden jackal (Canis aureus) cell, a gray wolf (Canis lupus) cell, a crab-eating fox (Cerdocyon thous) cell, a maned wolf (Chrysocyon brachyurus) cell, a dhole (Cuon alpinus) cell, a Falkland island wolf (Dusicyon australis) cell, a South American fox (Dusicyon avus) cell, a black-backed jackal (Lupulella mesomeias) cell, a side-striped j ackal (Lupulella adustus) cell, a culpeo (Lycalopex culpeo) cell, a Darwin’s fox (Lycalopex fulvipes) cell, a hoary fox (Lycalopex vetulus) cell, a pampas fox (Lycalopex gymnocercus) cell, a Sechuran fox (Lycalopex sechurae) cell, a South American gray fox (Lycalopex griseus) cell, or an African wild dog (Lycaon pictus) cell.

[0123] Also provided are transgenic animals comprising a recombinant host cell as described herein. In certain embodiments, the transgenic animal is a canine. In certain embodiments, the transgenic animal is a short-eared dog (Atelocynus microtis) an African wolf (Canis hipaster), a coyote (Canis latrans), a dog (Canis familiaris), an Ethiopian wolf (Canis simensis), a golden jackal (Canis aureus), a gray wolf (Canis lupus), a crab-eating fox (Cerdocyon thous), a maned wolf (Chrysocyon brachyurus), a dhole (Cuon alpinus), a Falkland island wolf (Dusicyon australis), a South American fox (Dusicyon avus), a black-backed jackal (Lupulella mesomeias), a side-striped jackal (Lupulella adustus), a culpeo (Lycalopex culpeo), a Darwin’s fox (Lycalopex fulvipes), a hoary fox (Lycalopex vetulus), a pampas fox (Lycalopex gymnocercus), a Sechuran fox (Lycalopex sechurae), a South American gray fox (Lycalopex griseus), or an African wild dog (Lycaon pictus).Transgenic animals

[0124] Provided herein are transgenic animals comprising at least one direwolf (Aenocyon dirus) gene variant associated with coat composition. The transgenic animals can, for example comprise at least one direwolf gene variant associated with coat compositions selected from the group consisting of a CBD103 variant, a FGF5 variant, a MC1R variant, a MFSD12 variant, an ASIP variant, a CORIN variant, a DOCK7 variant, a KRT71 variant, a KRT76 variant, a MITF variant, a MLPH variant, a MREG variant, and a TYRP1 variant. In certain embodiments, the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.

[0125] Also provided herein are transgenic animals comprising at least one direwolf (Aenocyon dirus) gene variant associated with vibrissae. The transgenic animal can, for example, comprise4908-6027-7382.1 31Attorney Docket No.: 069296.11210 / 28WO2at least one direwolf {Aenocyon dirus) gene variant associated with vibrissae selected from a CIB3 variant and a PMPCA variant.

[0126] In certain embodiments, the transgenic animal comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants. In some embodiments, the direwolf gene variant comprises one or more substitutions within the regulatory region and / or within the coding region of the gene as compared to a corresponding reference genome, wherein the corresponding reference genome is the genome of the animal prior to the one or more substitutions.

[0127] In certain embodiments, the transgenic animal fails to express an endogenous homologue of at least one of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA. In certain embodiments, the recombinant host cell fails to express the endogenous homologue of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0128] In certain embodiments, the transgenic animal is a canine. In certain embodiments, the transgenic animal is a short-eared dog {Atelocynus microtis), an African wolf {Canis lupaster), a coyote {Canis latrans), a dog {Canis familiaris), an Ethiopian wolf {Canis simensis), a golden jackal {Canis aureus), a gray wolf {Canis lupus), a crab-eating fox {Cerdocyon thous), a maned wolf {Chrysocyon brachyurus), a dhole {Cuon alpinus), a Falkland island wolf {Dusicyon australis), a South American fox {Dusicyon avus), a black-backed jackal {Lupulella mesomeias), a side-striped jackal {Lupulella adustus), a culpeo (Lycalopex culpeo), a Darwin’s fox {Lycalopex fulvipes), a hoary fox {Lycalopex vetulus), a pampas fox {Lycalopex gymnocercus), a Sechuran fox {Lycalopex sechurae), a South American gray fox {Lycalopex griseus), or an African wild dog {Lycao pictus).Methods for introducing direwolf gene variants into a cell

[0129] In certain embodiments of any of the aspects, the cell compositions described herein express a polypeptide encoded by the at least one isolated nucleic acid sequence having a direwolf gene variant nucleotide sequence.

[0130] The cells described herein can be transfected, contacted with, or administered an exogenous direwolf gene variant encoded by the isolated nucleic acids described herein by methods known in the art.4908-6027-7382.1 32Attorney Docket No.: 069296.11210 / 28WO2

[0131] In some embodiments, the at least one nucleic acid sequence encoding a direwolf gene variant is delivered via a vector.

[0132] A vector is a nucleic acid construct designed for delivery to a host cell or for transfer of genetic material between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer genetic material to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc.

[0133] In some embodiments of any of the aspects, the vector is selected from the group consisting of a plasmid, a cosmid, and a viral vector.

[0134] An expression vector is a vector that directs expression of an RNA or polypeptide (e.g., a direwolf polypeptide) from nucleic acid sequences contained therein linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell; a direwolf gene introduced to a viable cell is heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in animal cells for expression and in a prokaryotic host for cloning and amplification. “Expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification, and processing. “Expression products” include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene.

[0135] In some embodiments, a vector is capable of driving expression of one or more sequences in a mammalian cell; i.e., the vector is a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector’s control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et4908-6027-7382.1 33Attorney Docket No.: 069296.11210 / 28WO2al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.Methods of inhibiting or editing the expression of an endogenous gene

[0136] In some embodiments of any the aspects, the cell described herein does not express an endogenous homologue of the at least one direwolf gene variant described herein. In another embodiment of any of the aspects, the cell is edited to inhibit expression of an endogenous homologue of the at least one direwolf gene variant. In another embodiment of any of the aspects, the cell is edited to alter the regulatory and / or coding region to incorporate substitutions so that the endogenous homologue resembles the at least one direwolf gene variant.

[0137] In another embodiment of any of the aspects, the non-direwolf homologue of the exogenous nucleic acid sequence has been deleted or inactivated.

[0138] It is contemplated herein that when one or more direwolf gene variants are delivered to the host cell(s) it can be advantageous to modify the endogenous non-direwolf homologue of the one or more genes to render the endogenous gene or genes non-functional. It is further contemplated herein that if two or more direwolf genes are delivered to the host cell, one or both of the endogenous host cell genes would be altered. Thus, in this context, the host cell can comprise at least one non-functional endogenous homologue to the corresponding direwolf gene.

[0139] In the context of canine cells, the canine homologue(s) of the one or more direwolf genes to be expressed would be altered, deleted, or inhibited such that only the one or more direwolf gene(s) is(are) expressed by the cell. This can be achieved, for example, by standard gene editing of target sequences. It is also contemplated that rather than simply inactivating the endogenous gene, wholesale replacement of the endogenous gene, e. , via homologous recombination, or via selective editing of the non-direwolf homologue gene(s) to encode and express the direwolf variant gene sequence(s) could also be performed.

[0140] The target sequence can be determined by methods known in the art. For example, sequence alignment tools can be used to compare the direwolf nucleic acid sequences to those in the host organism, e.g., using NCBI Basic Local Alignment Sequence Tool (BLAST), OrthoMaM, Ensembl and / or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.4908-6027-7382.1 34Attorney Docket No.: 069296.11210 / 28WO2

[0141] Methods of inhibiting gene function in a host cell are known in the art. Non-limiting examples of gene knockdown, inhibition, and alteration include, e.g., gene editing enzymes, Transcription Activator-Like Effectors Nucleases (TALENS), inhibitory nucleic acids, and the like. Exemplary embodiments of types of inhibitory nucleic acids can include, e.g., siRNA, shRNA, miRNA, and / or a miRNA, which are known in the art. One of ordinary skill in the art can design and test an inhibitory agent that targets the endogenous homologue of a direwolf gene variant described herein.

[0142] Methods of preparing and delivering gene editing systems are described, e.g., in WO2015 / 013583 A2; US Pat. No. 10,640,789 B2; US Publication No. US2019 / 0367948 Al; US Publication No. 2017 / 0266320 Al; US Publication No. 2018 / 0171361 Al; US Publication No.2016 / 0175462 Al; and US Publication No. 2018 / 0195089 Al, the contents of each of which are incorporated herein by reference in their entirety.Methods of generating transgenic animals

[0143] This disclosure also provides for methods of making transgenic animals comprising at least one direwolf (Aenocyon dims) gene variant, wherein the direwolf gene variant is associated with a coat composition of a direwolf and / or a vibrissae of a direwolf.

[0144] In one embodiment of the disclosure, provided is a method of creating a transgenic animal comprising at least one direwolf (Aenocyon dims) gene variant, wherein the direwolf gene variant is associated with a coat composition of a direwolf and / or a vibrissae of a direwolf, the method comprising (a) obtaining a cell from the animal; (b) introducing into the cell at least one direwolf gene variant associated with coat composition and / or at least one direwolf gene variant associated with vibrissae; whereby introducing that at least one direwolf gene variant into the cell produces a recombinant cell; and (c) utilizing the recombinant cell to produce a transgenic animal; wherein the transgenic animal has the coat composition of a direwolf and / or the vibrissae of a direwolf.

[0145] In certain embodiments, the at least one direwolf (Aenocyon dims) gene variant associated with coat composition is selected from the group consisting of a CBD103 variant, a FGF5 variant, a MC1R variant, a MFSD12 variant, an ASIP variant, a CORIN variant, a DOCK7 variant, a KRT71 variant, a KRT76 variant, a MITF variant, a MLPH variant, a MREG variant, and a TYRP1 variant. In certain embodiments, the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.4908-6027-7382.1 3Attorney Docket No.: 069296.11210 / 28WO2

[0146] In certain embodiments, the at least one direwolf (Aenocyon dims) gene variant associated with vibrissae is selected from a CIB3 variant and a PMPCA variant.

[0147] In certain embodiments, the direwolf gene variant comprises at least one change in the nucleotide sequence of the gene. The change in the nucleotide sequence can, for example, be a substitution, an insertion, a deletion, or a combination thereof. In certain embodiments, the substitution, the insertion, the deletion, or a combination thereof is in a 5’ untranslated region of the gene, an intron of the gene, an exon of the gene, a 3’ untranslated region of the gene, or a combination thereof. The substitution, the insertion, the deletion, or a combination thereof can, for example, be in a regulatory region of the gene.

[0148] In certain embodiments, the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:53-65 and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122-134, and combinations thereof.

[0149] In certain embodiments, the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:66-67, and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-136, and combinations thereof.

[0150] In certain embodiments, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants are introduced into the cell.

[0151] In certain embodiments, the recombinant host cell fails to express an endogenous homologue of at least one of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA. In certain embodiments, the recombinant host cell fails to express the endogenous homologue of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0152] A variety of cells may be used in the methods of creating a transgenic animal. In one embodiment, the cell is a stem cell. The stem cell can, for example, be selected from an induced4908-6027-7382.1 36Attorney Docket No.: 069296.11210 / 28WO2stem cell, an embryonic stem (ES) cell, a mesenchymal stem cell (MSC), or combinations thereof. In an alternate embodiment, the cell is a reprogrammed cell. In yet another embodiment, the cell is a fibroblast cell or a mesenchymal cell. In a further embodiment, the cell is selected from the group consisting of a nerve cell, a cartilage cell, a bone cell, a muscle cell, a fat cell, and an epidermal cell. In another embodiment, the cell is an endothelial progenitor cell (EPC) or a pericyte.

[0153] In certain embodiments, the cell is a canine cell. In certain embodiments, the cell is a short-eared dog (Atelocynus microtis) cell, an African wolf (Canis lupaster) cell, a coyote (Cams latrans) cell, a dog (Canis familiaris) cell, an Ethiopian wolf (Canis simensis) cell, a golden jackal (Canis aureus) cell, a gray wolf (Canis lupus) cell, a crab-eating fox (Cerdocyon thous) cell, a maned wolf (Chrysocyon brachyurus) cell, a dhole (Cuon alpinus) cell, a Falkland island wolf (Dusicyon australis) cell, a South American fox (Dusicyon avus) cell, a black-backed jackal (Lupulella mesomeias) cell, a side-striped jackal (Lupulella adustus) cell, a culpeo (Lycalopex culpeo) cell, a Darwin’s fox (Lycalopex fulvipes) cell, a hoary fox (Lycalopex vetulus) cell, a pampas fox (Lycalopex gymnocercus) cell, a Sechuran fox (Lycalopex sechurae) cell, South American gray fox (Lycalopex griseus) cell, or an African wild dog (Lycao pictus) cell.

[0154] The disclosure also includes a transgenic animal made by these methods.EMBODIMENTS

[0155] The invention provides also the following non-limiting embodiments.

[0156] Embodiment 1 is a transgenic animal comprising at least one direwolf (Aenocyon dirus) gene variant associated with(a) coat compositions; and / or(b) vibrissae.

[0157] Embodiment 2 is the transgenic animal of embodiment 1, wherein the at least one direwolf (Aenocyon dirus) gene variant associated with coat composition is selected from the group consisting of a Beta-defensin 103 (CBD103) variant, a Fibroblast growth factor 5 (FGF5) variant, a Melanocortin receptor 1 (MC1R) variant, a Major facilitator superfamily domain containing 12 (MFSD12) variant, an Agouti signaling protein (ASIP) variant, a Serine peptidase (CORIN) variant, a Dedicator of cytokinesis 7 (DOCK7) variant, a Keratin 71 (KRT71) variant, a Keratin 76 (KRT76) variant, a Melanocyte inducing transcription factor (MITF) variant, a4908-6027-7382.1 37Attorney Docket No.: 069296.11210 / 28WO2Melanophilin (MLPH) variant, a Melanoregulin (MREG) variant, and a Tyrosinase related protein 1 (TYRP1) variant.

[0158] Embodiment 3 is the transgenic animal of embodiment 1, wherein the at least one direwolf (Aenocyon dints) gene variant associated with vibrissae is selected from a Calcium and integrin binding family member 3 (CIB3) variant and a Peptidase mitochondrial processing subunit alpha (PMPCA) variant.

[0159] Embodiment 4 is the transgenic animal of embodiment 2, wherein the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.

[0160] Embodiment 5 is the transgenic animal of embodiment 1, wherein the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene.

[0161] Embodiment 6 is the transgenic animal of embodiment 5, wherein the change in the nucleotide sequence is a substitution, an insertion, a deletion, or a combination thereof.

[0162] Embodiment 7 is the transgenic animal of embodiment 6, wherein the substitution, the insertion, the deletion, or a combination thereof is in a 5’ untranslated region of the gene, an intron of the gene, an exon of the gene, a 3’ untranslated region of the gene, or a combination thereof.

[0163] Embodiment 8 is the transgenic animal of embodiment 6, wherein the substitution, the insertion, the deletion, or a combination thereof is in a regulatory region of the gene.

[0164] Embodiment 9 is the transgenic animal of embodiment 2, wherein the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:53-65 and combinations thereof.

[0165] Embodiment 10 is the transgenic animal of embodiment 2, wherein the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122-134, and combinations thereof.

[0166] Embodiment 11 is the transgenic animal of embodiment 3, wherein the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:66-67, and combinations thereof.4908-6027-7382.1 38Attorney Docket No.: 069296.11210 / 28WO2

[0167] Embodiment 12 is the transgenic animal of embodiment 3, wherein the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-136, and combinations thereof.

[0168] Embodiment 13 is the transgenic animal of embodiment 1, wherein the transgenic animal comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants.

[0169] Embodiment 14 is the transgenic animal of any one of embodiments 1 to 13, wherein the transgenic animal fails to express an endogenous homologue of at least one of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0170] Embodiment 15 is the transgenic animal of embodiment 14, wherein the transgenic animal fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0171] Embodiment 16 is the transgenic animal of any one of embodiments 1 to 15, wherein the transgenic animal is a canine.

[0172] Embodiment 17 is the transgenic animal of embodiment 16, wherein the canine is selected from a short-eared dog (Atelocynus microtis), an African wolf (Canis lupaster), a coyote (Canis latrans), a dog (Canis familiaris), an Ethiopian wolf (Canis simensis), a golden jackal (Canis aureus), a gray wolf (Canis lupus), a crab-eating fox (Cerdocyon thous), a maned wolf (Chrysocyon brachyurus), a dhole (Cuon alpinus), a Falkland island wolf (Dusicyon australis), a South American fox Dusicyon avus), a black-backed jackal (Lupulella mesomeias), a side- striped j ackal (Lupulella adustus), a culpeo (Lycalopex culpeo), a Darwin’s fox (Lycalopex fulvipes), a hoary fox (Lycalopex vetulus), a pampas fox (Lycalopex gymnocercus), a Sechuran fox (Lycalopex sechurae), a South American gray fox (Lycalopex griseus), or an African wild dog (Lycaon pictus).

[0173] Embodiment 18 is a recombinant host cell comprising at least one direwolf (Aenocyon dir us) gene variant associated with(a) coat composition; and / or(b) vibrissae.

[0174] Embodiment 19 is the recombinant host cell of embodiment 18, wherein the at least one direwolf (Aenocyon dirus) gene variant associated with coat composition is selected from the4908-6027-7382.1 39Attorney Docket No.: 069296.11210 / 28WO2group consisting of a CBD103 variant, a FGF5 variant, a MC1R variant, a MFSD12 variant, an ASIP variant, a CORIN variant, a D0CK7 variant, a KRT71 variant, a KRT76 variant, a MITF variant, a MLPH variant, a MREG variant, and a TYRP1 variant.

[0175] Embodiment 20 is the recombinant host cell of embodiment 18, wherein the at least one direwolf (Aenocyon dirus) gene variant associated with vibrissae is selected from a CIB3 variant and a PMPCA variant.

[0176] Embodiment 21 is the recombinant host cell of embodiment 19, wherein the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.

[0177] Embodiment 22 is the recombinant host cell of embodiment 18, wherein the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene.

[0178] Embodiment 23 is the recombinant host cell of embodiment 22, wherein the change in the nucleotide sequence is a substitution, an insertion, a deletion, or a combination thereof.

[0179] Embodiment 24 is the recombinant host cell of embodiment 23, wherein the substitution, the insertion, the deletion, or a combination thereof is in a 5’ untranslated region of the gene, an intron of the gene, an exon of the gene, a 3’ untranslated region of the gene, or a combination thereof.

[0180] Embodiment 25 is the recombinant host cell of embodiment 23, wherein the substitution, the insertion, the deletion, or a combination thereof is in a regulatory region of the gene.

[0181] Embodiment 26 is the recombinant host cell of embodiment 19, wherein the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:53-65 and combinations thereof.

[0182] Embodiment 27 is the recombinant host cell of embodiment 19, wherein the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122-134, and combinations thereof.

[0183] Embodiment 28 is the recombinant host cell of embodiment 20, wherein the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:66-67, and combinations thereof.4908-6027-7382.1 40Attorney Docket No.: 069296.11210 / 28WO2

[0184] Embodiment 29 is the recombinant host cell of embodiment 20, wherein the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-136, and combinations thereof.

[0185] Embodiment 30 is the recombinant host cell of embodiment 18, wherein the recombinant host cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants.

[0186] Embodiment 31 is the recombinant host cell of any one of embodiments 18 to 30, wherein the recombinant host cell fails to express an endogenous homologue of at least one of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0187] Embodiment 32 is the recombinant host cell of embodiment 31, wherein the recombinant host cell fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0188] Embodiment 33 is the recombinant host cell of any one of embodiments 18 to 32, wherein the recombinant host cell is a canine cell.

[0189] Embodiment 34 is the recombinant host cell of embodiment 33, wherein the canine cell is selected from a short-eared dog (Atelocynus microtis) cell, an African wolf Cams lupaster) cell, a coyote (Cams latrans) cell, a dog (Cams familiaris) cell, an Ethiopian wolf (Cams simensis) cell, a golden jackal (Cams aureus) cell, a gray wolf (Cams lupus) cell, a crab-eating fox (Cerdocyon thous) cell, a maned wolf (Chrysocyon brachyurus) cell, a dhole (Cuon alpinus) cell, a Falkland island wolf (Dusicyon australis) cell, a South American fox (Dusicyon avus) cell, a black-backed jackal (Lupulella mesomeias) cell, a side-striped jackal (Lupulella adustus) cell, a culpeo (Lycalopex culpeo) cell, a Darwin’s fox (Lycalopex fulvipes) cell, a hoary fox (Lycalopex vetulus) cell, a pampas fox (Lycalopex gymnocercus) cell, a Sechuran fox (Lycalopex sechurae) cell, a South American gray fox (Lycalopex griseus) cell, or an African wild dog (Lycaon pictus) cell.

[0190] Embodiment 35 is the recombinant host cell of any one of embodiments 18 to 34, wherein the recombinant host cell is a stem cell.4908-6027-7382.1 41Attorney Docket No.: 069296.11210 / 28WO2

[0191] Embodiment 36 is the recombinant host cell of embodiment 35, wherein the stem cell is selected from an induced pluripotent stem cell (iPSC), an embryonic stem (ES) cell, or a mesenchymal stem cell (MSC).

[0192] Embodiment 37 is the recombinant host cell of any one of embodiments 18 to 34, wherein the recombinant host cell is a reprogrammed cell.

[0193] Embodiment 38 is the recombinant host cell of any one of embodiments 18 to 34, wherein the recombinant host cell is a fibroblast cell or a mesenchymal cell.

[0194] Embodiment 39 is the recombinant host cell of any one of embodiments 18 to 34, wherein the recombinant host cell is an endothelial progenitor cell (EPC) or a pericyte.

[0195] Embodiment 40 is the recombinant host cell of any one of embodiments 18 to 34, wherein the recombinant host cell is selected from the group consisting of a nerve cell, a cartilage cell, a bone cell, a muscle cell, a fat cell, and an epidermal cell.

[0196] Embodiment 41 is a method of creating a transgenic animal with a coat composition of a direwolf and / or a vibrissae of a direwolf, the method comprising(a) obtaining a cell from the animal;(b) introducing into the cell at least one direwolf gene variant associated with coat composition and / or at least one direwolf gene variant associated with vibrissae; whereby introducing that at least one direwolf gene variant into the cell produces a recombinant cell; and(c) utilizing the recombinant cell to produce a transgenic animal;wherein the transgenic animal has the coat composition of a direwolf and / or the vibrissae of a direwolf.

[0197] Embodiment 42 is the method of embodiment 41, wherein the at least one direwolf (Aenocyon dims) gene variant associated with coat composition is selected from the group consisting of a Beta-defensin 103 (CBD103) variant, a Fibroblast growth factor 5 (FGF5) variant, a Melanocortin receptor 1 (MC1R) variant, a Major facilitator superfamily domain containing 12 (MFSD12) variant, an Agouti signaling protein (ASIP) variant, a Serine peptidase (CORIN) variant, a Dedicator of cytokinesis 7 (DOCK7) variant, a Keratin 71 (KRT71) variant, a Keratin 76 (KRT76) variant, a Melanocyte inducing transcription factor (MITF) variant, a Melanophilin (MLPH) variant, a Melanoregulin (MREG) variant, and a Tyrosinase related protein 1 (TYRP1) variant.4908-6027-7382.1 42Attorney Docket No.: 069296.11210 / 28WO2

[0198] Embodiment 43 is the method of embodiment 41, wherein the at least one direwolf (Aenocyon dirus) gene variant associated with vibrissae is selected from a Calcium and integrin binding family member 3 (CIB3) variant and a Peptidase mitochondrial processing subunit alpha (PMPCA) variant.

[0199] Embodiment 44 is the method of embodiment 42, wherein the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.

[0200] Embodiment 45 is the method of embodiment 41, wherein the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene.

[0201] Embodiment 46 is the method of embodiment 45, wherein the change in the nucleotide sequence is a substitution, an insertion, a deletion, or a combination thereof.

[0202] Embodiment 47 is the method of embodiment 46, wherein the substitution, the insertion, the deletion, or a combination thereof is in a 5’ untranslated region of the gene, an intron of the gene, an exon of the gene, a 3’ untranslated region of the gene, or a combination thereof.

[0203] Embodiment 48 is the method of embodiment 46, wherein the substitution, the insertion, the deletion, or a combination thereof is in a regulatory region of the gene.

[0204] Embodiment 49 is the method of embodiment 42, wherein the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:53-65 and combinations thereof.

[0205] Embodiment 50 is the method of embodiment 42, wherein the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122-134, and combinations thereof.

[0206] Embodiment 51 is the method of embodiment 43, wherein the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:66-67, and combinations thereof.

[0207] Embodiment 52 is the method of embodiment 43, wherein the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-136, and combinations thereof.4908-6027-7382.1 43Attorney Docket No.: 069296.11210 / 28WO2

[0208] Embodiment 53 is the method of embodiment 41, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants are introduced into the cell.

[0209] Embodiment 54 is the method of any one of embodiments 41 to 53, wherein the transgenic animal fails to express an endogenous homologue of at least one of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0210] Embodiment 55 is the method of embodiment 54, wherein the transgenic animal fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

[0211] Embodiment 56 is the method of any one of embodiments 41-55, wherein the cell is a stem cell.

[0212] Embodiment 57 is the method of embodiment 56, wherein the stem cell is selected from an induced stem cell, an embryonic stem (ES) cell, or a mesenchymal stem cell (MSC).

[0213] Embodiment 58 is the method of any one of embodiments 41 to 55, wherein the cell is a reprogrammed cell.

[0214] Embodiment 59 is the method of any one of embodiments 41 to 55, wherein the cell is a fibroblast cell or a mesenchymal cell.

[0215] Embodiment 60 is the method of any one of embodiments 41 to 55, wherein the cell is selected from the group consisting of a nerve cell, a cartilage cell, a bone cell, a muscle cell, a fat cell, and an epidermal cell.

[0216] Embodiment 61 is the method of any one of embodiments 41 to 55, wherein the cell is an endothelial progenitor cell (EPC) or a pericyte.

[0217] Embodiment 62 is the method of any one of embodiments 41 to 55, wherein the animal is a canine.

[0218] Embodiment 63 is the method of embodiment 62, wherein the canine is selected from a short-eared dog (Atelocynus microtis), an African wolf (Canis lupaster), a coyote (Canis latrans), a dog (Canis familiaris), an Ethiopian wolf (Canis simensis), a golden jackal (Canis aureus), a gray wolf (Canis lupus), a crab-eating fox (Cerdocyon thous), a maned wolf (Chrysocyon brachyurus), a dhole (Cuon alpinus), a Falkland island wolf (Dusicyon australis), a South American fox (Dusicyon avus), a black-backed jackal (Lupulella mesomeias), a side-4908-6027-7382.1 44Attorney Docket No.: 069296.11210 / 28WO2striped jackal (Lupulella cidustus), a culpeo (lycalopex culpeo), a Darwin’s fox (lycalopex fulvipes) a hoary fox (Lycalopex vetulus) a pampas fox (Lycalopex gym nocercus)., a Sechuran fox (Lycalopex sechurae), a South American gray fox (Lycalopex griseus). or an African wild dog (Lycaon pictus).

[0219] Embodiment 64 is a transgenic animal made by the method of any one of embodiments 41 to 63.

[0220] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples, therefore, specifically point out the preferred embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.EXAMPLES

[0221] Example 1: Creation of Transgenic Animals comprising direwolf gene variants.

[0222] The animal work described herein has been approved and conducted under the oversight of an Institutional Animal Care and Use Committee (IACUC). Colossal uses the “Guide for the Care and Use of Laboratory Animals” when establishing animal research standards.

[0223] Materials and Methods:

[0224] Cell Engineering:

[0225] Blood-derived adherent endothelial progenitor cells (EPCs) and pericytes from wild and domestic canids represent a readily accessible, relatively non-invasive way to biobank living cells from animals (FIG. 5). Peripheral blood was collected by venipuncture from adult gray wolves, and the mononuclear fraction of cells separated from Red Blood Cells by centrifugation using a HISTOPAQUE® density gradient. The entire mononuclear fraction was plated in defined or commercial EPC medium (EGM™ or EGM™2 medium from Lonza; Basel, Switzerland) on plastic or collagen-coated cell culture plates or flasks and cultured overnight. The medium was changed the following day to remove all unattached cells. Within 5-10 days of culture, clusters of peripheral blood-derived, adherent cells were observed. These putative EPCs were reliably expanded, cryopreserved and reconstituted.

[0226] Genome Engineering:

[0227] As a proof of concept, five loci or genes of interest from Table 1 that are associated with core direwolf phenotypes were targeted. The genes are: LCORL, FGF5, MC1R, MFSD12, and4908-6027-7382.1 45Attorney Docket No.: 069296.11210 / 28WO2MSTN. LCORL is associated with size in domestic dogs, FGF5 is associated with coat composition across mammalian species, MC1R and MFSD12 are associated with coat composition and coloring across mammalian species, and MSTN is associated with muscle composition across mammalian species. CRISPR guides were designed for full gene knock-out (KO) in all 5 loci in gray wolf endothelial progenitor cell lines. Tested guides for each locus were assembled into a single 5X array and delivered to the gray wolf EPCs in a single transfection. Edited EPCs were sorted based on a GFP-reporter system. Monoclonal EPCs were established from the edited population and screened for editing efficiencies on the gene KOs of interest. Genotyping verified successful editing of intended targets with a combination of homozygous and heterozygous KOs. Edited EPCs were expanded and prepped for SCNT.

[0228] Somatic Cell Nuclear Transfer:

[0229] Blood-derived EPCs and pericytes from an adult male, gray wolf, Canis lupus (22CL04) were used as nuclear donors in enucleated domestic dog oocytes and activated by standard methods used in domestic dog cloning (Lee et al., “Dogs cloned from adult somatic cells,” Nature 436:641 (2005); Olsson et al., “Insights from one thousand cloned dogs,” Sci. Rep.12: 11209 (2022)). While canine SCNT is routinely performed with fibroblasts, the successful cloning from adherent, blood-derived, non-hematopoietic cells has not been reported to date. After overnight in vitro culture, cleavage to the 2-cell stage is evident in some of the iSCNT gray wolf embryos-indicative of effective activation (FIG. 6).

[0230] Analysis:

[0231] Both 1-cell and 2-cell iSCNT embryos reconstructed with genetically engineered gray wolf EPCs were surgically transferred into the oviducts of domestic dog recipients in natural estrus. Uterine implantation was reliably detected by trans-abdominal ultrasound (US) as early as day 20 in embryo transfer (ET) recipients, with cardiac development and active beating observed by Day 23 using trans-abdominal ultrasound (US) (FIG. 7).

[0232] Transabdominal radiography and micro-Computed Tomography (microCT) were used to detect normal bone formation between Day 42-47 of post-implantation development of iSCNT genetically engineered gray wolf fetuses.

[0233] Cells lines and tissue were isolated from iSCNT genetically engineered wolf fetuses. Post-implantation iSCNT wildtype and gene-edited gray wolf fetuses were isolated after ovariohysterectomy between 23 to 45 days after oviductal embryo transfer. In one experiment,4908-6027-7382.1 46Attorney Docket No.: 069296.11210 / 28WO2ET of 34 iSCNT genetically engineered gray wolf embryos resulted in the implantation of 6 fetuses. A timed-ovariohysterectomy (OHE) was performed at Day 33 after embryo transfer, and fetuses were isolated from the uterus under sterile conditions. Tissues were collected from these genetically engineered fetuses for downstream transcriptomic analysis and comparisons in the genes of interest.

[0234] Transfer of 339 embryos split between 8 recipient dogs resulted in 4 pregnancies of gene-edited iSCNT gray wolf cloned embryos with two singleton pregnancies going to term. Two living gene-edited, male wolf pups were delivered by c-section at day 62 and 63 post embryo transfer. Genotyping of the umbilical cords from each pup demonstrated that both were derived from the same clonal gene-edited EPC line. Both pups demonstrated phenotypic changes from the wildtype Agouti coat color in the starting adult donor wolf to pure white by the disruption of both the MC1R and MFSD12 loci preventing the production of eumelanin and pheomelanin. Additional phenotypic and transcriptomic analysis, such as cranio-facial and whole body measurements, among others, will continue as the pups grow.

[0235] Birth and development of gene-edited direwolves

[0236] On October 1, 2024, two male Colossal Dire Wolves were born, and on January 30, 2025, version 2 of the Colossal Dire Wolf was bom. All three exhibited phenotypes corresponding to the implemented edits and matched the anticipated characteristics. These visible traits are not present in traditional, non-edited grey wolves. The observed physical traits provided the first confirmation of the genetic modifications and aligned with the expected outcomes.

[0237] Over the past year, all three specimens have grown and expressed the physical changes that were selected during the editing process. As they have matured into the sub-adult stage, all three display normal exotic canid behaviors and have achieved the major developmental milestones anticipated for behavioral progression. Photographs of the development of the dire wolves are shown in FIGs. 9A-9F.

[0238] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description.4908-6027-7382.1 47

Claims

Atorney Docket No.: 069296.11210 / 28WO2CLAIMSIt is claimed:

1. A transgenic animal comprising at least one direwolf (Aenocyon dirus) gene variant associated with(a) coat compositions; and / or(b) vibrissae.

2. The transgenic animal of claim 1, wherein the at least one direwolf (Aenocyon dirus) gene variant associated with coat composition is selected from the group consisting of a Beta-defensin 103 (CBD103) variant, a Fibroblast growth factor 5 (FGF5) variant, a Melanocortin receptor 1 (MC1R) variant, a Major facilitator superfamily domain containing 12 (MFSD12) variant, an Agouti signaling protein (ASIP) variant, a Serine peptidase (CORIN) variant, a Dedicator of cytokinesis 7 (DOCK7) variant, a Keratin 71 (KRT71) variant, a Keratin 76 (KRT76) variant, a Melanocyte inducing transcription factor (MITF) variant, a Melanophilin (MLPH) variant, a Melanoregulin (MREG) variant, and a Tyrosinase related protein 1 (TYRP1) variant.

3. The transgenic animal of claim 1, wherein the at least one direwolf Aenocyon dirus) gene variant associated with vibrissae is selected from a Calcium and integrin binding family member 3 (CIB3) variant and a Peptidase mitochondrial processing subunit alpha (PMPCA) variant.

4. The transgenic animal of claim 2, wherein the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.

5. The transgenic animal of claim 1, wherein the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene.

6. The transgenic animal of claim 5, wherein the change in the nucleotide sequence is a substitution, an insertion, a deletion, or a combination thereof.

7. The transgenic animal of claim 6, wherein the substitution, the insertion, the deletion, or a combination thereof is in a 5’ untranslated region of the gene, an intron of the gene, an exon of the gene, a 3’ untranslated region of the gene, or a combination thereof.

8. The transgenic animal of claim 6, wherein the substitution, the insertion, the deletion, or a combination thereof is in a regulatory region of the gene.4908-6027-7382.1Atorney Docket No.: 069296.11210 / 28WO29. The transgenic animal of claim 2, wherein the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 53-65 and combinations thereof.

10. The transgenic animal of claim 2, wherein the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122-134, and combinations thereof.

11. The transgenic animal of claim 3, wherein the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:66-67, and combinations thereof.

12. The transgenic animal of claim 3, wherein the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-136, and combinations thereof.

13. The transgenic animal of claim 1, wherein the transgenic animal comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants.

14. The transgenic animal of any one of claims 1 to 13, wherein the transgenic animal fails to express an endogenous homologue of at least one of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

15. The transgenic animal of claim 14, wherein the transgenic animal fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

16. The transgenic animal of any one of claims 1 to 15, wherein the transgenic animal is a canine.

17. The transgenic animal of claim 16, wherein the canine is selected from a short-eared dog (Atelocynus microtis), an African wolf (Canis lupaster), a coyote (Canis latrans), a dog (Canis familiaris), an Ethiopian wolf (Canis simensis), a golden jackal (Canis aureus), a gray wolf (Canis lupus), a crab-eating fox (Cerdocyon thous), a maned wolf (Chrysocyon brachyurus), a dhole (Cuon alpinus), a Falkland island wolf (Dusicyon australis), a South American fox (Dusicyon avus), a black-backed jackal (Lupulella mesomeias), a side-striped jackal (Lupulella4908-6027-7382.1 49Atorney Docket No.: 069296.11210 / 28WO2adustus), a culpeo (Lycalopex culpeo), a Darwin’s fox (lycalopex fulvipes), a hoary fox (Lycalopex vetulus), a pampas fox (Lycalopex gymnocercus), a Sechuran fox (Lycalopex sechurae), a South American gray fox (Lycalopex griseus), or an African wild dog (Lycaon pictus).

18. A recombinant host cell comprising at least one direwolf (Aenocyon dirus) gene variant associated with(a) coat composition; and / or(b) vibrissae.

19. The recombinant host cell of claim 18, wherein the at least one direwolf (Aenocyon dirus) gene variant associated with coat composition is selected from the group consisting of a CBD103 variant, a FGF5 variant, a MC1R variant, a MFSD12 variant, an ASIP variant, a CORIN variant, a DOCK7 variant, a KRT71 variant, a KRT76 variant, a MITF variant, a MLPH variant, a MREG variant, and a TYRP1 variant.

20. The recombinant host cell of claim 18, wherein the at least one direwolf (Aenocyon dirus gene variant associated with vibrissae is selected from a CIB3 variant and a PMPCA variant.

21. The recombinant host cell of claim 19, wherein the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.

22. The recombinant host cell of claim 18, wherein the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene.

23. The recombinant host cell of claim 22, wherein the change in the nucleotide sequence is a substitution, an insertion, a deletion, or a combination thereof.

24. The recombinant host cell of claim 23, wherein the substitution, the insertion, the deletion, or a combination thereof is in a 5’ untranslated region of the gene, an intron of the gene, an exon of the gene, a 3’ untranslated region of the gene, or a combination thereof.

25. The recombinant host cell of claim 23, wherein the substitution, the insertion, the deletion, or a combination thereof is in a regulatory region of the gene.

26. The recombinant host cell of claim 19, wherein the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 53-65 and combinations thereof.4908-6027-7382.1 50Atorney Docket No.: 069296.11210 / 28WO227. The recombinant host cell of claim 19, wherein the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122-134, and combinations thereof.

28. The recombinant host cell of claim 20, wherein the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:66-67, and combinations thereof.

29. The recombinant host cell of claim 20, wherein the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-136, and combinations thereof.

30. The recombinant host cell of claim 18, wherein the recombinant host cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants.

31. The recombinant host cell of any one of claims 18 to 30, wherein the recombinant host cell fails to express an endogenous homologue of at least one of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

32. The recombinant host cell of claim 31, wherein the recombinant host cell fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA33. The recombinant host cell of any one of claims 18 to 32, wherein the recombinant host cell is a canine cell.

34. The recombinant host cell of claim 33, wherein the canine cell is selected from a shorteared dog Atelocynus microtis) cell, an African wolf (Canis lupaster) cell, a coyote (Canis latrans) cell, a dog (Canis familiaris) cell, an Ethiopian wolf (Canis simensis) cell, a golden jackal (Canis aureus) cell, a gray wolf (Canis lupus) cell, a crab-eating fox (Cerdocyon thous) cell, a maned wolf (Chrysocyon brachyurus) cell, a dhole (Cuon alpinus) cell, a Falkland island wolf (Dusicyon australis) cell, a South American fox (Dusicyon avus) cell, a black-backed jackal (Lupulella mesomeias) cell, a side-striped jackal (Lupulella adustus) cell, a culpeo (Lycalopex culpeo) cell, a Darwin’s fox Lycalopex fulvipes) cell, a hoary fox (Lycalopex vetulus) cell, a4908-6027-7382.1 51Atorney Docket No.: 069296.11210 / 28WO2pampas fox (Lycalopex gymnocercus) cell, a Sechuran fox (lycalopex sechurae) cell, a South American gray fox (Lycalopex griseus) cell, or an African wild dog Lycaon pictus) cell.

35. The recombinant host cell of any one of claims 18 to 34, wherein the recombinant host cell is a stem cell.

36. The recombinant host cell of claim 34, wherein the stem cell is selected from an induced pluripotent stem cell (iPSC), an embryonic stem (ES) cell, or a mesenchymal stem cell (MSC).

37. The recombinant host cell of any one of claims 18 to 34, wherein the recombinant host cell is a reprogrammed cell.

38. The recombinant host cell of any one of claims 18 to 34, wherein the recombinant host cell is a fibroblast cell or a mesenchymal cell.

39. The recombinant host cell of any one of claims 18 to 34, wherein the recombinant host cell is an endothelial progenitor cell (EPC) or a pericyte.

40. The recombinant host cell of any one of claims 18 to 34, wherein the recombinant host cell is selected from the group consisting of a nerve cell, a cartilage cell, a bone cell, a muscle cell, a fat cell, and an epidermal cell.

41. A method of creating a transgenic animal with a coat composition of a direwolf and / or a vibrissae of a direwolf, the method comprising(a) obtaining a cell from the animal;(b) introducing into the cell at least one direwolf gene variant associated with coat composition and / or at least one direwolf gene variant associated with vibrissae; whereby introducing that at least one direwolf gene variant into the cell produces a recombinant cell; and(c) utilizing the recombinant cell to produce a transgenic animal;wherein the transgenic animal has the coat composition of a direwolf and / or the vibrissae of a direwolf.

42. The method of claim 41, wherein the at least one direwolf (Aenocyon dints) gene variant associated with coat composition is selected from the group consisting of a Beta-defensin 103 (CBD103) variant, a Fibroblast growth factor 5 (FGF5) variant, a Melanocortin receptor 1 (MC1R) variant, a Major facilitator superfamily domain containing 12 (MFSD12) variant, an Agouti signaling protein (ASIP) variant, a Serine peptidase (CORIN) variant, a Dedicator of cytokinesis 7 (DOCK7) variant, a Keratin 71 (KRT71) variant, a Keratin 76 (KRT76) variant, a4908-6027-7382.1 52Atorney Docket No.: 069296.11210 / 28WO2Melanocyte inducing transcription factor (MITF) variant, a Melanophilin (MLPH) variant, a Melanoregulin (MREG) variant, and a Tyrosinase related protein 1 (TYRP1) variant.

43. The method of claim 41, wherein the at least one direwolf (Aenocyon dims) gene variant associated with vibrissae is selected from a Calcium and integrin binding family member 3 (CIB3) variant and a Peptidase mitochondrial processing subunit alpha (PMPCA) variant.

44. The method of claim 42, wherein the CBD103 variant, the FGF5 variant, the MC1R variant, the MFSD12 variant, and combinations thereof are loss-of-function variants.

45. The method of claim 41, wherein the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene.

46. The method of claim 45, wherein the change in the nucleotide sequence is a substitution, an insertion, a deletion, or a combination thereof.

47. The method of claim 46, wherein the substitution, the insertion, the deletion, or a combination thereof is in a 5’ untranslated region of the gene, an intron of the gene, an exon of the gene, a 3’ untranslated region of the gene, or a combination thereof.

48. The method of claim 46, wherein the substitution, the insertion, the deletion, or a combination thereof is in a regulatory region of the gene.

49. The method of claim 42, wherein the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs:53-65 and combinations thereof.

50. The method of claim 42, wherein the at least one direwolf gene variant associated with coat composition comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 122-134, and combinations thereof.

51. The method of claim 43, wherein the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 66-67, and combinations thereof.

52. The method of claim 43, wherein the at least one direwolf gene variant associated with vibrissae comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 135-136, and combinations thereof.4908-6027-7382.1 53Atorney Docket No.: 069296.11210 / 28WO253. The method of claim 41, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 direwolf gene variants are introduced into the cell.

54. The method of any one of claims 41 to 53, wherein the transgenic animal fails to express an endogenous homologue of at least one of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA.

55. The method of claim 54, wherein the transgenic animal fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of CBD103, FGF5, MC1R, MFSD12, ASIP, CORIN, DOCK7, KRT71, KRT76, MITF, MLPH, MREG, TYRP1, CIB3, and PMPCA 56. The method of any one of claims 41-55, wherein the cell is a stem cell.

57. The method of claim 16, wherein the stem cell is selected from an induced stem cell, an embryonic stem (ES) cell, or a mesenchymal stem cell (MSC).

58. The method of any one of claims 41-55, wherein the cell is a reprogrammed cell.

59. The method of any one of claims 41-55, wherein the cell is a fibroblast cell or a mesenchymal cell.

60. The method of any one of claims 41-55, wherein the cell is selected from the group consisting of a nerve cell, a cartilage cell, a bone cell, a muscle cell, a fat cell, and an epidermal cell.

61. The method of any one of claims 41-55, wherein the cell is an endothelial progenitor cell (EPC) or a pericyte.

62. The method of any one of claims 41-55, wherein the animal is a canine.

63. The method of claim 62, wherein the canine is selected from a short-eared dog (Atelocynus microtis), an African wolf (Canis hipaster), a coyote (Canis latrans), a dog (Canis familiaris), an Ethiopian wolf (Canis simensis), a golden jackal (Canis aureus , a gray wolf (Canis lupus), a crab-eating fox (Cerdocyon thous), a maned wolf (Chrysocyon brachyurus), a dhole (Cuon alpinus), a Falkland island wolf (Dusicyon australis), a South American fox (Dusicyon avus), a black-backed jackal (Lupulella mesomeias), a side-striped jackal (Lupulella adustus), a culpeo (Lycalopex culpeo), a Darwin’s fox (Lycalopex fulvipes), a hoary fox (Lycalopex vetulus), a pampas fox (Lycalopex gymnocercus), a Sechuran fox (Lycalopex sechurae), a South American gray fox (Lycalopex griseus), or an African wild dog (Lycaon pictus).

64. A transgenic animal made by the method of any one of claims 41-63.4908-6027-7382.1 54

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