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 body size, muscle mass, and / or metabolism; craniofacial morphology; and / or neurobehavior. Also provided are methods of making recombinant cells and / or transgenic animals that comprise at least one direwolf (Aenocyon dirus) gene variant associated with body size, muscle mass, and / or metabolism; craniofacial morphology; and / or neurobehavior.

Description

Atorney Docket No.: 069296.11209 / 28WO1TRANSGENIC 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 / 746,773, filed on January 17, 2025. and U.S. Provisional Application No. 63 / 746,786, 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, uses thereof, and methods of making the same. In particular, the disclosure relates to gene edited, and / or reprogrammed mammalian cells containing direwolf (Aenocyon dims) 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 (069296.28WO1 Sequence Lisitng.xml; size: 14,819.769 bytes; and creation date of January 5, 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 deextinction 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.

[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 body size, muscle mass, and / or metabolism of direwolves; craniofacial features of direwolves; and neurobehavior of direwolves.4937-9365-5942 1Attorney Docket No.: 069296.11209 / 28WO1BRIEF SUMMARY OF THE INVENTION

[0006] Provided herein are transgenic animals comprising at least one direwolf (Aenocyon dims) gene variant associated with (a) body size, muscle mass, and / or metabolism; (b) craniofacial morphology; and / or (c) neurobehavior.

[0007] In certain embodiments, the at least one direwolf (Aenocyon dims) gene variant associated with body size, muscle mass, and / or metabolism is selected from the group consisting of an ADAM metallopeptidase with thrombospondin type 1 motif 9 (ADAMTS9) variant, a Ligand dependent nuclear receptor corepressor like (LCORL) variant, a Myostatin (MSTN) variant, a Ligand dependent nuclear receptor corepressor like and chromosome condensation protein G (LCORL -NCAPG) variant, an A disintegrin and metallopeptidase domain 10 (ADAM 10) variant, an Apolipoprotein H (APOH) variant, a Branched chain keto acid dehydrogenase El subunit beta (BCKDHB) variant, a BRCA1 associated protein (BRAP) variant, a Coiled-coil-helix-coiled-coil-helix domain containing 5 (CHCHD5) variant, a Muscarinic acetylcholine receptor M3 (CHRM3) variant, a Carnitine palmitoyltransferase 1C (CPT1C) variant, aDrebrin 1 (DBN1) variant, an Emendymin related 1 (EPDR1) variant, an Ethanolamine-phosphate phospholyase (ETNPPL) variant, a Fatty acid binding protein 4 (FABP4) variant, a High mobility group AT-hook 2 (HMGA2) variant, a Kelch repeat and BTB domain containing 13 (KBTBD13) variant, a Low-density7lipoprotein receptor (LDLR) variant, a Mitogen-activated protein kinase kinase 5 (MAP2K5) variant, a Nuclear receptor corepressor 1 (NCOR1) variant, a Neurotrophic receptor tyrosine kinase 3 (NTRK3) variant, a Phosphoenolpyruvate carboxykinase 1 (PCK1) variant, a Pleckstrin homology domain containing SI (PLEKHS1) variant, a Pancreatic lipase related protein 1 (PNLIPRP1) variant, aPPFIA binding protein 1 (PPFIBP1) variant, a Solute carrier family 30 member 8 (SLC30A8) variant, a ST3 beta-galactoside-alpha-2.3 -sialytransferase 2 (ST3GAL2) variant, a Syntaxin 16 (STX16) variant, a WNT inhibitory factor 1 (WIFI) variant, a Wingless type MMTV integration site family member 2 (WNT2) variant, a WW domain containing oxidoreductase (WWOX) variant, and aZinc finger protein FOG family member 2 (ZFPM2) variant. In certain embodiments, the ADAMTS9 variant, the LCORL variant, the MSTN variant, and combinations thereof are loss-of-function variants.

[0008] In certain embodiments, the at least one direwolf (Aenocyon dims) gene variant associated with craniofacial morphology is selected from the group consisting of an Ataxin 10 (ATXN10) variant, a Calcium voltage-gated channel subunit alpha IS (CACNA1S) variant, a Capping actin protein of muscle Z-line subunit beta (CAPZB) variant, a Cadherin 24 (CDH24) variant, a Distal-less homeobox 3 (DLX3) variant, a Grow th arrest specific 64937-9365-5942.1 2Attorney Docket No.: 069296.11209 / 28WO1(GAS6) variant, a G protein coupled receptor kinase 1 (GRK1) variant, a High mobility' group AT-hook 2 (HMGA2) variant, a Microtubule associated protein RP / EB family member 1 (MAPRE1) variant, a Microtubule associated protein RP / EB family member 2 (MAPRE2) variant, a Melanin concentrating hormone receptor 1 (MCHR1) variant, a DAN family BMP antagonist (NBL1) variant, an Odd-skipped related transcription factor 1 (OSR1) variant, a RNA polymerase I subunit A (P0LR1A) variant, a Proteasome subunit beta 11 (PSMB11) variant, a Pentatricopeptide repeat domain 3 (PTCD3) variant, a SIK family kinase 3 (SIK3) variant, a Transmembrane and coiled-coil domain family 1 (TMCC1) variant, a Transmembrane protein 255B (TMEM25B) variant, a X-prolyl aminopeptidase 3 (XPNPEP3) variant, a WNT inhibitory' factor 1 (WIFI) variant, and aZFP36 ring finger protein like 1 (ZFP36L1) variant.

[0009] In certain embodiments, the at least one direwolf (Aenocyon citrus) gene variant associated with neurobehavior is selected from aNeurexophilin 2 (NXPH2) variant and a Slit guidance ligand 1 (SLIT2) variant.

[0010] 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.

[0011] In certain embodiments, the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism 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: 1-32 and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 70- 101, and combinations thereof.

[0012] In certain embodiments, the at least one direwolf gene variant associated with craniofacial morphology 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: 16, 29, 33-52, and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with craniofacial morphology comprises a4937-9365-5942.1 3Atorney Docket No.: 069296.11209 / 28WO1nucleotide sequence selected from the group consisting of SEQ ID NOs:85, 98, 102-121, and combinations thereof.

[0013] In certain embodiments, the at least one direwolf gene variant associated with neurobehavior 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:68-69 and combinations thereof. In certain embodiments, the at least one direwolf gene vanant associated with neurobehavior comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 137-138 and combinations thereof.

[0014] In certain embodiments, the transgenic animal comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) direwolf gene variants.

[0015] In certain embodiments, the transgenic animal fails to express an endogenous homologue of at least one of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAM 10, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24. DLX3. GAS6, GRK1. MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2. In certain embodiments, the transgenic animal fails to express the endogenous homologue of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15. 20, 25, 30, 35, 40, 45, 50, 51, 52. 53, 54 (or any number in between) of ADAMTS9, LCORL. MSTN, LCORL-NCAPG. ADAMI 0, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10. CACNA1S, CAPZB, CDH24. DLX3. GAS6, GRK1, MAPRE1, MAPRE2, MCHRL NBLl, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2.

[0016] 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 (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 hrachyurus), 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), a4937-9365-5942.1 4Atorney Docket No.: 069296.11209 / 28WO1pampas fox (Lycctlopex gymnocercus), a Sechuran fox (Lycalopex sechurae), a South American gray fox (Lycalopex griseus). or an African wild dog (Lycaon pictus).

[0017] Also provided herein are recombinant host cells compnsing at least one direwolf (Aenocyon dints) gene variant associated with (a) body size, muscle mass, and / or metabolism; (b) craniofacial morphology; and / or (c) neurobehavior.

[0018] In certain embodiments, the at least one direwolf (Aenocyon dirus) gene variant associated with body size, muscle mass, and / or metabolism is selected from the group consisting of an ADAMTS9 variant, a LCORL variant, a MSTN variant, a LCORL-NC APG variant, an ADAMI 0 variant, an APOH variant, a BCKDHB variant, a BRAP variant, a CHCHD5 variant, a CHRM3 variant, a CPT1C variant, aDBNl variant, an EPDR1 variant, an ETNPPL variant, a FABP4 variant, a HMGA2 variant, a KBTBD13 variant, a LDLR variant, aMAP2K5 variant, aNCORl variant, aNTRK.3 variant, a PCKl variant, a PLEKHS1 variant, aPNLIPRPl variant, a PPFIBP1 variant, a SLC30A8 variant, a ST3GAL2 variant, a STX16 variant, a WIFI variant, a WNT2 variant, a WWOX variant, and aZFPM2 variant. In certain embodiments, the ADAMTS9 variant, the LCORL variant, the MSTN variant, and combinations thereof are loss-of-function variants.

[0019] In certain embodiments, the at least one direwolf (Aenocyon dirus) gene variant associated with craniofacial morphology is selected from the group consisting of an ATXN10 variant, a CACNA1S variant, a CAPZB variant, a CDH24 variant, a DLX3 variant, a GAS6 variant, a GRK1 variant, a HMGA2 variant, a MAPRE1 variant, a MAPRE2 variant, a MCHR1 variant, aNBLl variant, an OSR1 variant, a POLR1 A variant, a PSMBl l variant, a PTCD3 variant, a SIK3 variant, a TMCC1 variant, a TMEM25B variant, a WIFI variant, a XPNPEP3 variant, and aZFP36Ll variant.

[0020] In certain embodiments, the at least one direwolf (Aenocyon dirus) gene variant associated with neurobehavior is selected from aNXPH2 variant and a SLIT2 variant.

[0021] 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.

[0022] In certain embodiments, the direwolf gene variant associated with body size, muscle mass, and / or metabolism comprises a nucleotide sequence having at least 80%, at least 85%,4937-9365-5942.1 5Attorney Docket No.: 069296.11209 / 28WO1at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-32 and combinations thereof. In certain embodiments, the direwolf gene variant associated with body size, muscle mass, and / or metabolism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:70-101, and combinations thereof.

[0023] In certain embodiments, the direwolf gene variant associated with craniofacial morphology 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: 16, 29, 33-52, and combinations thereof. In certain embodiments, the direwolf gene variant associated with craniofacial morphology comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:85. 98. 102-121. and combinations thereof.

[0024] In certain embodiments, the direwolf gene variant associated with neurobehavior 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: 68-69 and combinations thereof. In certain embodiments, the direwolf gene variant associated with neurobehavior comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 137-138 and combinations thereof.

[0025] In certain embodiments, the recombinant host cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15. 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) direwolf gene variants.

[0026] In certain embodiments, the recombinant host cell fails to express an endogenous homologue of at least one of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAMI 0, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1. NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2. 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, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54 (or any number in between) of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAM10, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1. PNLIPRP1, PPFIBP1. SLC30A8, ST3GAL2. STX16, WIFI. WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1,4937-9365-5942.1 6Attorney Docket No.: 069296.11209 / 28WO1MAPRE2, MCHR1, NBL1, 0SR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2. and SLIT2.

[0027] In certain embodiments, the recombinant host cell is a canine cell. The canine cell can, for example, be selected from 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.

[0028] 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).

[0029] In certain embodiments, the recombinant host cell is a reprogrammed cell.

[0030] 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.

[0031] 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 body size, muscle mass, and / or metabolism of a direwolf; a craniofacial morphology of a direwolf; and / or a neurobehavior of a direwolf.

[0032] 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 body size, muscle mass, and / or metabolism; at least one direwolf gene variant associated with craniofacial morphology; and / or at least one direwolf gene variant associated with neurobehavior; 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 body size, muscle mass, and / or metabolism of a direwolf; the craniofacial morphology of a direwolf; and / or the neurobehavior of a direwolf.4937-9365-5942.1 7Atorney Docket No.: 069296.11209 / 28WO1

[0033] In certain embodiments, the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism is selected from the group consisting of an ADAMTS9 variant, a LCORL variant, a MSTN variant, a LCORL-NCAPG variant, an ADAM 10 variant, an APOH variant, a BCKDHB variant, a BRAP variant, a CHCHD5 variant, a CHRM3 variant, a CPT1C variant, a DBN1 variant, an EPDR1 variant, an ETNPPL variant, a FABP4 variant, a HMGA2 variant, a KBTBD13 variant, a LDLR variant, a MAP2K5 variant, a NCOR1 variant, aNTRK3 variant, a PCKl variant, aPLEKHSl variant, a PNLIPRPl variant, a PPFIBP1 variant, a SLC30A8 variant, a ST3GAL2 variant, a STX16 variant, a WIFI variant, a WNT2 variant, WWOX variant, and a ZFPM2 variant. In certain embodiments, the ADAMTS9 variant, the LCORL variant, the MSTN variant, and combinations thereof are loss-of-function variants.

[0034] In certain embodiments, the at least one direwolf (Aenocyon citrus) gene variant associated with craniofacial morphology is selected from the group consisting of an ATXN10 variant, a CACNA1S variant, a CAPZB variant, a CDH24 variant, a DLX3 variant, a GAS6 variant, a GRK1 variant, aHMGA2 variant. aMAPREl variant, a MAPRE2 variant, a MCHR1 variant, aNBLl variant, an OSR1 variant, aPOLRIA variant, a PSMBll variant, a PTCD3 variant, a SIK3 variant, a TMCC1 variant, a TMEM25B variant, a XPNPEP3 variant, a WIFI variant, and aZFP36Ll variant.

[0035] In certain embodiments, the at least one direwolf (Aenocyon dims) gene variant associated with neurobehavior is selected from aNXPH2 variant and a SLIT2 variant.

[0036] 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 regulator}' region of the gene.

[0037] In certain embodiments, the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism 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: 1-32 and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:70-101, and combinations thereof.4937-9365-5942.1 8Atorney Docket No.: 069296.11209 / 28WO1

[0038] In certain embodiments, the at least one direwolf gene variant associated with craniofacial morphology 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: 16, 29, 33-52, and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with craniofacial morphology7comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:85, 98, 102-121, and combinations thereof.

[0039] In certain embodiments, the at least one direwolf gene variant associated with neurobehavior 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:68-69 and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with neurobehavior comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 137-138 and combinations thereof.

[0040] In certain embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) direwolf gene variants are introduced into the cell.

[0041] In certain embodiments, the recombinant cell fails to express an endogenous homologue of at least one of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAMI 0, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2. In certain embodiments, the recombinant cell fails to express the endogenous homologue of 2, 3, 4, 5. 6, 7, 8. 9, 10, 15. 20. 25. 30. 35, 40, 45, 50, 51, 52, 53, 54 (or any number in between) of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAMIO, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1. PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI. WNT2, WWOX, ZFPM2, ATXN10. CACNA1S, CAPZB, CDH24. DLX3. GAS6. GRK1. MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2.

[0042] 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).4937-9365-5942.1 9Attorney Docket No.: 069296.11209 / 28WO1

[0043] In certain embodiments, the recombinant cell is a reprogrammed cell.

[0044] 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.

[0045] 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 (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).

[0046] Also provided are transgenic animals made by the methods disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0047] 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.

[0048] FIG. 1 show s 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 aligned sequences 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 aFigTree (Ramabaut, FigTree v 1.3.1, Institute of Evolutionary' Biology, University of Edinburgh: tree.bio.ed.ac.uk / software / figtree / (2010)) method as used for generating plot.

[0049] 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 Famham Sebastopol Tokyo: O’Reilly (2020)) methods were used for calling sequence variants;4937-9365-5942.1 10Attorney Docket No.: 069296.11209 / 28WO1bcftools (Danecek et al., Gigascience 10:giab008 (2021)) methods were used for variant processing to assess sequence similarity; Rtidyverse (Wickham et al., J. Open Source Softw 4:1686 (2019)) methods were used for generating plot.

[0050] 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 tidy verse (Wickham et al., J. Open Source Softw 4:1686 (2019)) methods were used for generating plot.

[0051] FIG. 4 shows the amino acid and 3D structural comparison between proteins encoded by two genes of interest, MITF and LCORL, in both gray w olf 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.

[0052] FIG. 5 shows images of adherent endothelial progenitor cells (EPCs) (left) and pericytes (right) derived from peripheral blood of adult gray wolves (Cants lupus).

[0053] FIG. 6 shows an image of cleavage of genetically engineered gray wolf embryos produced by interspecies somatic cell nuclear transfer (iSCNT) with EPCs.

[0054] FIG. 7 shows an image of an ultrasound providing confirmation of a successful pregnancy with a transgenic wolf fetus.

[0055] FIG. 8 shows an image of a heatmap of 50 of the most variable genes between unedited and genetically edited w olf 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 26:139-1404937-9365-5942.1 11Atorney Docket No.: 069296.11209 / 28WO1(2010)), DESeq2 (Love et al., Genome Biol. 15:550 (2014)), and IsoformSwitchAnalyzeR (Viting-Seerup and Sandelin, Bioinformatics 35:4469-4471 (2019)) methods were used for detecting differentially expressed genes and transcripts; and the R package clusterProfiler (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).

[0056] 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

[0057] This disclosure is based on the discover}' that using only a minimal number of direwolf (Aenocyon dims) gene variants it is possible to generate a transgenic animal with the body size, muscle mass, and / or metabolism of a direwolf; the craniofacial morphology of a direwolf; and / or the neurobehavior of a direwolf. Accordingly, the disclosure provides methods of generating transgenic animals by introducing at least one direwolf (Aenocyon dims) gene variant associated with (a) body size, muscle mass, and / or metabolism; (b) craniofacial morphology; and / or (c) neurobehavior into the animal. The at least one direwolf (Aenocyon dims) gene variant associated with associated with body size, muscle mass, and / or metabolism is selected from the group consisting of an ADAMTS9 variant, a LCORL variant, a MSTN variant, a LCORL-NCAPG variant, an ADAM10 variant, an APOH variant, a BCKDHB variant, a BRAP variant, a CHCHD5 variant, a CHRM3 variant, a CPT1C variant, a DBN1 variant, an EPDR1 variant, an ETNPPL variant, a FABP4 variant, a HMGA2 variant, a KBTBD13 variant, a LDLR variant, a MAP2K5 variant, aNCORl variant, aNTRK3 variant, a PCKl variant, a PLEKHSl variant, a PNLIPRPl variant, a PPFIBP1 variant, a SLC30A8 variant, a ST3GAL2 variant, a STX16 variant, a WIFI variant, a WNT2 variant, a WWOX variant, and a ZFPM2 variant. The at least one direwolf (Aenocyon dims) gene variant associated with craniofacial morphology is selected from the group consisting of an ATXN10 variant, a CACNA1S variant, a CAPZB variant, a CDH24 variant, a DLX3 variant, a GAS6 variant, a GRK1 variant, a HMGA2 variant, a MAPRE1 variant, aMAPRE2 variant, a MCHRl variant, aNBLl variant, an OSR1 variant, aPOLRIA variant, a PSMB11 variant, a PTCD3 variant, a SIK.3 variant, a TMCC1 variant, a TMEM25B variant, a WIFI variant, aXPNPEP3 variant, and aZFP36Ll variant. The at4937-9365-5942.1 12Attorney Docket No.: 069296.11209 / 28WO1least one direwolf (Aenocyon dims gene variant associated with neurobehavior is selected from a NXPH2 variant and a SLIT2 variant.

[0058] 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.

[0059] 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

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary7skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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 other4937-9365-5942.1 13Attorney Docket No.: 069296.11209 / 28WO1integer 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, “of’ 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).

[0065] 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.”

[0066] 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.

[0067] 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.

[0068] The words “right,” “left,” “lower,” and “upper” designate directions in the drawings to which reference is made.

[0069] 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 include4937-9365-5942.1 14Attorney Docket No.: 069296.11209 / 28WO1variations 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.

[0070] 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.

[0071] 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.

[0072] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appt. 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, Wl), 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)).

[0073] 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: 3389-3402, 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 word4937-9365-5942.1 15Atorney Docket No.: 069296.11209 / 28WO1hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0074] 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 negativescoring 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)).

[0075] 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 similarity7provided 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.

[0076] 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, a polypeptide 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.

[0077] 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.4937-9365-5942.1 16Atorney Docket No.: 069296.11209 / 28WO1“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.

[0078] 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.

[0079] 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 ty pe of cell, e.g., a primary7cell, 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 of prokary otic, eukaryotic, or archaeal cell. In some instances, the host cell is a bacterial cell. In some instances, the host cell is a mammalian cell. In some embodiments, the mammalian host cell is a canine cell.

[0080] 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.

[0081] 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.4937-9365-5942.1 17Attorney Docket No.: 069296.11209 / 28WO1The 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, acety lation, phosphory lation, 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.

[0082] 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.

[0083] 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.

[0084] 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-stable inheritance. Host cells comprising the transformed nucleic acid fragment are referred to as “recombinant” or “transgenic” or “transformed” organisms.

[0085] 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.

[0086] As used herein, “gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.4937-9365-5942.1 18Atorney Docket No.: 069296.11209 / 28WO1

[0087] 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.

[0088] 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.

[0089] As used herein, the phrase “body size, muscle mass, and / or metabolism” refers to the increase in body size, muscle mass, and / or metabolism in the transgenic animal comprising the at least one direwolf gene variant, which would resemble the body size, muscle mass, and / or metabolism of a direwolf. The increase in body size, muscle mass, and / or metabolism can be measured in comparison to a control, e.g., by measuring the body size, muscle mass, and / or metabolism in the same animal without the at least one direwolf gene variant and comparing it to the transgenic animal comprising the at least one direwolf gene variant.

[0090] As used herein, the phrase “craniofacial morphology ” refers to the structure of the face and skull of the transgenic animal comprising the at least one direwolf gene variant. The face and skull of the transgenic animal comprising the at least one direwolf gene variant would resemble the face and skull of a direwolf. The difference in the craniofacial morphology can be determined in comparison to a control, e.g., by measuring the features of the face and skull in the same animal without the at least one direwolf gene variant and comparing it to the transgenic animal comprising the at least one direwolf gene variant.

[0091] As used herein, the phrase “neurobehavior” refers to the relationship between the brain and nervous system and how it affects behavior, emotion, and learning in the transgenic animal comprising the at least one direwolf gene variant. The neurobehavior of the transgenic animal comprising the at least one direwolf gene variant would resemble the neurobehavior of a direwolf. The difference in neurobehavior can be determined in comparison to a control, e.g., by measuring the neurobehavior of the same animal without the4937-9365-5942.1 19Atorney Docket No.: 069296.11209 / 28WO1at least one direwolf gene variant and comparing it to the transgenic animal comprising the at least one direwolf gene variant.

[0092] 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.

[0093] 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 of morphological 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 other4937-9365-5942.1 20Atorney Docket No.: 069296.11209 / 28WO1morphological or structural characteristics. In one embodiment, the marker is a cell surface marker.

[0094] 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.g. , 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.

[0095] 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, AZA, dexamethasone, VP A, TSA, SAHA, PD0325901 + CHIR99021(2i) and A-83-01. In some embodiments, the reprogramming genes or factors are Oct4, Klf4, SOX2, and c-Myc.

[0096] 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,4937-9365-5942.1 21Atorney Docket No.: 069296.11209 / 28WO1for example, by introducing or expressing exogenous polypeptide factors. Reprogramming is not typically observed under native conditions in vivo or in vitro.

[0097] 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.

[0098] 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 Variants

[0099] 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 deextinction 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 body size, muscle mass, and / or metabolism similar to direwolves; craniofacial morphology similar to direwolves; and / or neurobehavior 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 will4937-9365-5942.1 22Attorney Docket No.: 069296.11209 / 28WO1advance synthetic biology and will serve as a precedent for advanced multiplexed editing in organisms.

[0100] The isolated nucleic acids, vectors, recombinant cells, and transgenic animals described herein are based, in part, on the discover}’ that cells (e.g., a short-eared dog (Atelocynus microtis) cell, an African wolf (Canis hipaster) 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 dirus). 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 direw olf genes can be made to viable cells of other, nonhuman 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 synthetic alternative to wildlife products and new tools for understanding genetic diversity and cellular biology in endangered and extinct species of wildlife.

[0101] 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 direw olf 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 “direw olf 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.4937-9365-5942.1 23Attorney Docket No.: 069296.11209 / 28WO1In 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 regulator ' 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.

[0102] 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 (wvw.daiw-intreeoflife.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 Cams lupus. Cants lupus familiaris, and Aenocyon dirus. where Cants lupus is used as the reference genome. 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 phenoty pe-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 phenoty pe-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 wolf4937-9365-5942.1 24Atorney Docket No.: 069296.11209 / 28WO1genome 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 regulator}7regions 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”).

[0103] 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 DNA sequences not well-evidenced from ancient DNA sequencing data, which will be left as extant gray wolf sequence.

[0104] Thus, provided herein are isolated nucleic acid sequences comprising at least one direwolf (Aenocyon dims) gene variant associated with (a) body size, muscle mass, and / or metabolism; (b) craniofacial morphology7; and / or (c) neurobehavior. The isolated nucleic acid sequences can, for example, comprise at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism selected from an ADAMTS9 variant, a LCORL variant, a MSTN variant, a LCORL-NCAPG variant, an ADAM10 variant, an APOH variant, a BCKDHB variant, a BRAP variant, a CHCHD5 variant, a CHRM3 variant, a CPT1C variant, a DBN1 variant, an EPDR1 variant, an ETNPPL variant, a FABP4 variant, a HMGA2 variant. aKBTBD13 variant, aLDER variant, aMAP2K5 variant, aNCORl variant, aNTRK3 variant, aPCKl variant, a PLEKHS l variant, a PNLIPRPl variant, a PPFIBP1 variant, a SLC30A8 variant, a ST3GAL2 variant, a STX16 variant, a WIFI variant, a WNT2 variant, a WWOX variant, and a ZFPM2 variant. In certain embodiments, the ADAMTS9 variant, the LCORE variant, the MSTN variant, and combinations thereof are loss-of-function variants.4937-9365-5942.1 25Attorney Docket No.: 069296.11209 / 28WO1

[0105] Also provided are isolated nucleic acid sequences comprising at least one direwolf (Aenocyon dims) gene variant associated with craniofacial morphology selected from the group consisting of an ATXN10 variant, a CACNA1S variant, a CAPZB variant, a CDH24 variant, a DLX3 variant, a GAS6 variant, a GRK1 variant, a HMGA2 variant, a MAPRE1 variant, aMAPRE2 variant, a MCHRl variant, aNBLl variant, an OSR1 variant, aPOLRIA variant, a PSMB11 variant, a PTCD3 variant, a SIK3 variant, a TMCC1 variant, a TMEM25B variant, a WIFI variant, aXPNPEP3 variant, and aZFP36Ll variant.

[0106] Also provided are isolated nucleic acid sequences comprising at least one direwolf (Aenocyon dims) gene variant associated with neurobehavior selected from a NXPH2 variant and a SLIT2 variant.

[0107] 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.

[0108] 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 to about 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.

[0109] In certain embodiments, the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism 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: 1-32 and combinations thereof. The at least one direwolf4937-9365-5942.1 26Atorney Docket No.: 069296.11209 / 28WO1gene variant associated with body size, muscle mass, and / or metabolism 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: 1-32 and combinations thereof. The at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism can, for example, comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs:70-101 and combinations thereof.

[0110] In certain embodiments, the at least one direwolf gene variant associated with craniofacial morphology 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: 16. 29. 33-52 and combinations thereof. The at least one direwolf gene variant associated with craniofacial morphology 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: 16, 29, 33-52. and combinations thereof. The at least one direwolf gene variant associated with craniofacial morphology can, for example, comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs:85, 98, 102-121, and combinations thereof.

[0111] In certain embodiments, the at least one direwolf gene variant associated with neurobehavior 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:68-69 and combinations thereof. The at least one direwolf gene variant associated with neurobehavior 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:68-69 and combinations thereof. The at least one direyvolf gene variant associated with neurobehavior can, for example, comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 137-138 and combinations thereof.

[0112] In certain embodiments, the isolated nucleic acids comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) direwolf gene variants.

[0113] 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,4937-9365-5942.1 27Atorney Docket No.: 069296.11209 / 28WO1or more than one substitution, as compared to the canine (e.g., wolf) sequence. Thus, by way of an example, a direwolf gene variant for ADAM 10 can. for example, comprise 1, 5, 10, 15, 20, 30, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 360, 370, 380, 388 (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. By way of another example, a direwolf gene variant for ATXN10 can, for example, compose 1, 5, 10, 15, 20, 30, 40, 45, 50. 75. 100, 150, 200. 250, 300. 350, 400. 410, 420, 427 (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 1.4937-9365-5942.1 28Atorney Docket No.: 069296.11209 / 28WO14937-9365-5942.1 29Atorney Docket No.: 069296.11209 / 28WO1Total Variants in Gene can include variants in introns, exons, and regulatory regions.

[0114] Also provided are isolated vectors comprising the isolated nucleic acid sequences described herein.

[0115] Also provided are recombinant host cells comprising at least one direwolf (Aenocyon dints)' gene variant associated with (a) body size, muscle mass, and / or metabolism; (b) craniofacial morphology7; and / or (c) neurobehavior.

[0116] In certain embodiments, the at least one direwolf (Aenocyon dints) gene variant associated with body size, muscle mass, and / or metabolism is selected from the group consisting of an ADAMTS9 variant, a LCORL variant, a MSTN variant, an LCROL-NCAPG variant, an ADAMI 0 variant, an APOH variant, a BCKDHB variant, a BRAP variant, a CHCHD5 variant, a CHRM3 variant, a CPT1C variant, aDBNl variant, an EPDR1 variant, an ETNPPL variant, a FABP4 variant, a HMGA2 variant, a KBTBD13 variant, a LDLR4937-9365-5942.1 30Atorney Docket No.: 069296.11209 / 28WO1variant, a MAP2K5 variant, a NC0R1 variant, a NTRK3 variant, a PCK1 variant, a PLEKHS1 variant. aPNLIPRPl variant, a PPFIBPl variant, a SLC30A8 variant, a ST3GAL2 variant, a STX16 variant, a WIFI variant, a WNT2 variant, a WWOX variant, and a ZFPM2 variant. In certain embodiments, the ADAMTS9 variant, the LCORL variant, the MSTN variant, and combinations thereof are loss-of-function variants.

[0117] In certain embodiments, the at least one direwolf (Aenocyon dims) gene variant associated with craniofacial morphology is selected from the group consisting of an ATXN10 variant, a CACNA1S variant, a CAPZB variant, a CDH24 variant, a DLX3 variant, a GAS6 variant, a GRK1 variant, aHMGA2 variant, aMAPREl variant, a MAPRE2 variant, a MCHR1 variant, aNBLl variant, an OSR1 variant, aPOLRIA variant, a PSMBll variant, a PTCD3 variant, a SIK3 variant, a TMCC1 variant, a TMEM25B variant, a WIFI variant, a XPNPEP3 variant, and aZFP36Ll variant.

[0118] In certain embodiments, the at least one direwolf (Aenocyon dims) gene variant associated with neurobehavior is selected from aNXPH2 variant and a SLIT2 variant.

[0119] In certain embodiments, the recombinant host cell comprises 2, 3, 4, 5, 6, 7, 8. 9, 10, 15, 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) direwolf gene variants. In some embodiments, the direwolf gene variant comprises one or more substitutions within the regulator}7region 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.

[0120] In certain embodiments, the recombinant host cell fails to express an endogenous homologue of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAMI 0, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCORL NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2. In certain embodiments, the recombinant host cell fails to express the endogenous homologue of 2, 3, 4. 5. 6, 7, 8. 9, 10, 15. 20. 25. 30. 35. 40. 45, 50, 51, 52, 53, 54 (or any number in between) of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAM10, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1. PNLIPRP1, PPFIBP1. SLC30A8, ST3GAL2. STX16, WIFI. WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1,4937-9365-5942.1 31Attorney Docket No.: 069296.11209 / 28WO1MAPRE2, MCHR1, NBL1, 0SR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2. and SLIT2.

[0121] 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, 15, 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) of the isolated nucleic acids described herein.

[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, 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, a South American gray fox (Lycalopex griseus) cell, or an African wild dog (Lycaon pictus) cell.

[0123] The direwolf gene variants described herein can be used in any combination to be expressed in any recombinant host cell as described herein.Cells

[0124] 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 eukary otic 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, the cell 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.

[0125] 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.4937-9365-5942.1 32Attorney Docket No.: 069296.11209 / 28WO1

[0126] 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 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 w olf (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)).

[0127] 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.

[0128] The cells described herein can be from any tissue isolated from an organism by methods know n 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, skin cells, 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.

[0129] 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 renew7themselves through4937-9365-5942.1 33Atorney Docket No.: 069296.11209 / 28WO1mitotic cell division and can differentiate into more specialized cell ty pes. 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.

[0130] 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.

[0131] 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 ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAM10, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16. WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1 S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2. 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, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54 (or any number in between) of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAMI 0, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCKL PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8. ST3GAL2. STX16, WIFI. WNT2, WWOX, ZFPM2. ATXN10, CACNA1S. CAPZB.CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2.

[0132] 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,4937-9365-5942.1 34Attorney Docket No.: 069296.11209 / 28WO1an Ethiopian wolf (Cams simensis) cell, a golden jackal (Canis aureus) cell, a gray wolf (Cants 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 gr is eus) cell, or an African wild dog (Lycaon pictus) cell.

[0133] 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 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).Transgenic animals

[0134] Provided herein are transgenic animals comprising at least one direwolP (Aenocyon dims) gene variant associated with body size, muscle mass, and / or metabolism. The transgenic animals can, for example comprise at least one direw-olf gene variant associated with body size, muscle mass, and / or metabolism selected from the group consisting of an ADAMTS9 variant, a LCORL variant, a MSTN variant, a LCORL-NCAPG variant, an ADAMI 0 variant, an APOH variant, a BCKDHB variant, a BRAP variant, a CHCHD5 variant, a CHRM3 variant, a CPT1C variant, a DBN1 variant, an EPDR1 variant, an ETNPPL variant, a FABP4 variant, a HMGA2 variant, a KBTBD13 variant, a LDLR variant, aMAP2K5 variant, aNCORI variant, aNTRK3 variant, aPCKl variant, a PLEKHS l variant, a PNLIPRP1 variant, a PPFIBP1 variant, a SLC30A8 variant, a ST3GAL2 variant, a STX16 variant, a WIFI variant, a WNT2 variant, a WWOX variant, and aZFPM2 variant. In certain embodiments, the ADAMTS9 variant, the LCORL variant, the MSTN variant, and combinations thereof are loss-of-function variants.4937-9365-5942.1 35Atorney Docket No.: 069296.11209 / 28WO1

[0135] Also provided herein are transgenic animals comprising at least one direwolf (Aenocyon dims) gene variant associated with craniofacial morphology. The transgenic animal can, for example, comprise at least one direwolf gene variant associated with craniofacial morphology selected from the group consisting of an ATXN10 variant, a CACNA1S variant, a CAPZB variant, a CDH24 variant, a DLX3 variant, a GAS6 variant, a GRK1 variant, a HMGA2 variant, a MAPRE1 variant, a MAPRE2 variant, a MCHR1 variant, aNBLl variant, an OSR1 variant, aPOLRIA variant, a PSMBll variant, a PTCD3 variant, a SIK3 variant, aTMCCl variant, aTMEM25B variant, a WIFI variant, aXPNPEP3 variant, and aZFP36Ll variant.

[0136] Also provided herein are transgenic animals comprising at least one direwolf (Aenocyon dims) gene variant associated with neurobehavior. The transgenic animal can, for example comprise at least one direwolf gene variant associated with neurobehavior selected from a NXPH2 variant and a SLIT2 variant.

[0137] In certain embodiments, the transgenic animal comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) 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.

[0138] In certain embodiments, the transgenic animal fails to express an endogenous homologue of at least one of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAMI 0, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2. 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, 15. 20, 25, 30, 35, 40, 45, 50, 51, 52. 53. 54 (or any number in between) of ADAMTS9, LCORL. MSTN, LCORL-NCAPG. ADAM10, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1. PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10. CACNA1S, CAPZB, CDH24. DLX3. GAS6. GRK1. MAPRE1,4937-9365-5942.1 36Attorney Docket No.: 069296.11209 / 28WO1MAPRE2, MCHR1, NBL1, 0SR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2. and SLIT2.

[0139] 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 (Lycaon pictus).Methods for introducing direwolf gene variants into a cell

[0140] 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 vanant nucleotide sequence.

[0141] 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.

[0142] In some embodiments, the at least one nucleic acid sequence encoding a direwolf gene variant is delivered via a vector.

[0143] 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.

[0144] 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.

[0145] 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, for4937-9365-5942.1 37Atorney Docket No.: 069296.11209 / 28WO1example, 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.

[0146] 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, et al., 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

[0147] 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.

[0148] In another embodiment of any of the aspects, the non-direwolf homologue of the exogenous nucleic acid sequence has been deleted or inactivated.

[0149] 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 cell4937-9365-5942.1 38Atorney Docket No.: 069296.11209 / 28WO1can comprise at least one non-functional endogenous homologue to the corresponding direwolf gene.

[0150] 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.g, 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.

[0151] 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.

[0152] 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.

[0153] 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

[0154] This disclosure also provides for methods of making transgenic animals comprising at least one direwolf (Aenocyon dints) gene variant, wherein the direwolf gene variant is associated with a body size, muscle mass, and / or metabolism of a direwolf; a craniofacial morphology of a direwolf; and / or a neurobehavior of a direwolf.

[0155] 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 direwolf4937-9365-5942.1 39Atorney Docket No.: 069296.11209 / 28WO1gene variant is associated with a body size, muscle mass, and / or metabolism of a direwolf; a craniofacial morphology of a direwolf; and / or a neurobehavior 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 body size, muscle mass, and / or metabolism; at least one direwolf gene variant associated with craniofacial morphology; and / or at least one direwolf gene variant associated with neurobehavior; 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 body size, muscle mass, and / or metabolism of a direwolf; the craniofacial morphology7of a direwolf; and / or the neurobehavior of a direwolf.

[0156] In certain embodiments, the at least one direwolf (Aenocyon citrus) gene variant associated with body size, muscle mass, and / or metabolism is selected from the group consisting of an ADAMTS9 variant, a LCORL variant, a MSTN variant, an LCROL-NCAPG variant, an ADAMI 0 variant, an APOH variant, a BCKDHB variant, a BRAP variant, a CHCHD5 variant, a CHRM3 variant, a CPT1C variant, a DBN1 variant, an EPDR1 variant, an ETNPPL variant, a FABP4 variant, a HMGA2 variant, a KBTBD13 variant, a LDLR variant, a MAP2K5 variant, a NCOR1 variant, a NTRK3 variant, a PCK1 variant, a PLEKHS1 variant, aPNLIPRPl variant, a PPFIBP1 variant, a SLC30A8 variant, a ST3GAL2 variant, a STX16 variant, a WIFI variant, a WNT2 variant, a WWOX variant, and a ZFPM2 variant. In certain embodiments, the ADAMTS9 variant, the LCORL variant, the MSTN variant, and combinations thereof are loss-of-function variants.

[0157] In certain embodiments, the at least one direwolf (Aenocyon dims) gene variant associated with craniofacial morphology is selected from the group consisting of an ATXN10 variant, a CACNA1S variant, a CAPZB variant, a CDH24 variant, a DLX3 variant, a GAS6 variant, a GRK1 variant, a HMGA2 variant, a MAPRE1 variant, a MAPRE2 variant, a MCHR1 variant, aNBLl variant, an OSR1 variant, a POLR1A variant, a PSMB11 variant, a PTCD3 variant, a SIK3 variant, a TMCC1 variant, a TMEM25B variant, a WIFI variant, a XPNPEP3 variant, and aZFP36Ll variant.

[0158] In certain embodiments, the at least one direwolf (Aenocyon dims) gene variant associated with neurobehavior is selected from aNXPH2 variant and a SLIT2 variant.

[0159] 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 5’4937-9365-5942.1 40Atorney Docket No.: 069296.11209 / 28WO1untranslated 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.

[0160] In certain embodiments, the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism 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: 1-32 and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:70-101, and combinations thereof.

[0161] In certain embodiments, the at least one direwolf gene variant associated with craniofacial morphology 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: 16, 29, 33-52, and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with craniofacial morphology comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:85, 98, 102-121, and combinations thereof.

[0162] In certain embodiments, the at least one direwolf gene variant associated with neurobehavior 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:68-69 and combinations thereof. In certain embodiments, the at least one direwolf gene variant associated with neurobehavior comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 137-138 and combinations thereof.

[0163] In certain embodiments, the at least 2, 3, 4, 5, 6, 7, 8. 9, 10, 15, 20, 25. 30. 40. 50, 51, 52, 53, 54 (or any number in between) direwolf gene variants are introduced into the cell.

[0164] In certain embodiments, the recombinant cell fails to express an endogenous homologue of at least one of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAMI 0, APOH. BCKDHB. BRAP. CHCHD5, CHRM3. CPT1C, DBN1, EPDR1, ETNPPL, FABP4. HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1. NTRK3. PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2. In certain embodiments, the recombinant cell fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53,4937-9365-5942.1 41Atorney Docket No.: 069296.11209 / 28WO154 (or any number in between) of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAM10, APOH. BCKDHB. BRAP. CHCHD5, CHRM3, CPT1C, DBN1, EPDR1.ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2. and SLIT2.

[0165] 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 induced stem 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.

[0166] 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 hipcister) 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 aviis) cell, a black-backed j ackal (Lupulella mesomeias) cell, a side-striped jackal (Lupiilella 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 (Lycaon pictus) cell.

[0167] The disclosure also includes a transgenic animal made by these methods.EMBODIMENTS

[0168] The invention provides also the following non-limiting embodiments.

[0169] Embodiment l is a transgenic animal comprising at least one direwolf (Aenocyon dims) gene variant associated with(a) body size, muscle mass, and / or metabolism;(b) craniofacial morphology: and / or(c) neurobehavior.4937-9365-5942.1 42Atorney Docket No.: 069296.11209 / 28WO1

[0170] Embodiment 2 is the transgenic animal of embodiment 1, wherein the at least one direwolf (Aenocyon dints)' gene variant associated with body size, muscle mass, and / or metabolism is selected from the group consisting of an ADAM metallopeptidase with thrombospondin type 1 motif 9 (ADAMTS9) variant, a Ligand dependent nuclear receptor corepressor like (LCORL) variant, a Myostatin (MSTN) variant, a Ligand dependent nuclear receptor corepressor like and chromosome condensation protein G (LCORL-NCAPG) variant, an A disintegrin and metallopeptidase domain 10 (ADAM 10) variant, an Apolipoprotein H (APOH) variant, a Branched chain keto acid dehydrogenase El subunit beta (BCKDHB) variant, a BRCA1 associated protein (BRAP) variant, a Coiled-coil-helix-coiled-coil-helix domain containing 5 (CHCHD5) variant, a Muscarinic acetylcholine receptor M3 (CHRM3) variant, a Carnitine palmitoyltransferase 1C (CPT1C) variant, a Drebrin 1 (DBN1) variant, an Emendymin related 1 (EPDR1) variant, an Ethanolaminephosphate phospholyase (ETNPPL) variant, a Fatty acid binding protein 4 (FABP4) variant, a High mobility group AT-hook 2 (HMGA2) variant, a Kelch repeat and BTB domain containing 13 (KBTBD13) variant, a Low-density lipoprotein receptor (LDLR) variant, a Mitogen-activated protein kinase kinase 5 (MAP2K5) variant, a Nuclear receptor corepressor 1 (NCOR1) variant, a Neurotrophic receptor tyrosine kinase 3 (NTRK3) variant, a Phosphoenolpyruvate carboxykinase 1 (PCK1) variant, a Pleckstrin homology domain containing SI (PLEKHS1) variant, a Pancreatic lipase related protein 1 (PNLIPRP1) variant, a PPFIA binding protein 1 (PPFIBP1) variant, a Solute carrier family 30 member 8 (SLC30A8) variant, a ST3 beta-galactoside-alpha-2,3-sialytransferase 2 (ST3GAL2) variant, a Syntaxin 16 (STX16) variant, a WNT inhibitory factor 1 (WIFI) variant, a Wingless type MMTV integration site family member 2 (WNT2) variant, a WW domain containing oxidoreductase (WWOX) variant, and aZinc finger protein FOG family member 2 (ZFPM2) variant.

[0171] Embodiment 3 is the transgenic animal of embodiment 1 or 2, wherein the at least one direwolf (Aenocyon dirus) gene variant associated with craniofacial morphology is selected from the group consisting of an Ataxin 10 (ATXN10) variant, a Calcium voltagegated channel subunit alpha IS (CACNA1S) variant, a Capping actin protein of muscle Z-line subunit beta (CAPZB) variant, a Cadherin 24 (CDH24) variant, a Distal-less homeobox 3 (DLX3) variant, a Grow th arrest specific 6 (GAS6) variant, a G protein coupled receptor kinase 1 (GRK1) variant, a High mobility group AT-hook 2 (HMGA2) variant, a Microtubule associated protein RP / EB family member 1 (MAPRE1) variant, a Microtubule associated protein RP / EB family member 2 (MAPRE2) variant, a Melanin concentrating hormone4937-9365-5942.1 43Atorney Docket No.: 069296.11209 / 28WO1receptor 1 (MCHR1) variant, a DAN family BMP antagonist (NBL1) variant, an Oddskipped related transcription factor 1 (OSR1) variant, a RNA polymerase I subunit A (P0LR1A) variant, a Proteasome subunit beta 11 (PSMB11) variant, a Pentatricopeptide repeat domain 3 (PTCD3) variant, a SIK family kinase 3 (SIK3) variant, a Transmembrane and coiled-coil domain family 1 (TMCC1) variant, a Transmembrane protein 255B (TMEM25B) variant, a X-prolyl aminopeptidase 3 (XPNPEP3) variant, a WNT inhibitory factor 1 (WIFI) variant, and aZFP36 ring finger protein like 1 (ZFP36L1) variant.

[0172] Embodiment 4 is the transgenic animal of any one of embodiments 1-3, wherein the at least one direwolf (Aenocyon dims) gene variant associated with neurobehavior is selected from aNeurexophilin 2 (NXPH2) variant and a Slit guidance ligand 1 (SLIT2) variant.

[0173] Embodiment 5 is the transgenic animal of embodiment 2, wherein the ADAMTS9 variant, the LCORL variant, and / or the MSTN variant are loss-of-function variants.

[0174] Embodiment 6 is the transgenic animal of any one of embodiments 1-5, wherein the direwolf gene variant comprises at least one change in the nucleotide sequence of the gene.

[0175] Embodiment 7 is the transgenic animal of embodiment 6, wherein the change in the nucleotide sequence is a substitution, an insertion, a deletion, or a combination thereof.

[0176] Embodiment 8 is the transgenic animal of embodiment 7, 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.

[0177] Embodiment 9 is the transgenic animal of embodiment 7, wherein the substitution, the insertion, the deletion, or a combination thereof is in a regulatory region of the gene.

[0178] Embodiment 10 is the transgenic animal of embodiment 2, wherein the direwolf gene variant associated with body size, muscle mass, and / or metabolism 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: 1-32 and combinations thereof.

[0179] Embodiment 11 is the transgenic animal of embodiment 2, wherein the direwolf gene variant associated with body size, muscle mass, and / or metabolism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:70-101, and combinations thereof.

[0180] Embodiment 12 is the transgenic animal of embodiment 3, wherein the direwolf gene variant associated with craniofacial morphology comprises a nucleotide sequence having at4937-9365-5942.1 44Atorney Docket No.: 069296.11209 / 28WO1least 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:16, 29, 33-52. and combinations thereof.

[0181] Embodiment 13 is the transgenic animal of embodiment 3, wherein the direwolf gene variant associated with craniofacial morphology comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:85, 98, 102-121, and combinations thereof.

[0182] Embodiment 14 is the transgenic animal of embodiment 4, wherein the direwolf gene variant associated with neurobehavior 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: 68-69 and combinations thereof.

[0183] Embodiment 15 is the transgenic animal of embodiment 4, wherein the direwolf gene variant associated with neurobehavior comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 137-138 and combinations thereof.

[0184] Embodiment 16 is the transgenic animal of embodiment 1, wherein the transgenic animal comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) direwolf gene variants.

[0185] Embodiment 17 is the transgenic animal of any one of embodiments 1 to 16, wherein the transgenic animal fails to express an endogenous homologue of at least one of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAMI 0, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2. KBTBD13, LDLR. MAP2K5, NCOR1, NTRK3, PCK1. PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8. ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1 S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2.

[0186] Embodiment 18 is the transgenic animal of embodiment 17, wherein the transgenic animal fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54 (or any number in between) of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAM10, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16. WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2.

[0187] Embodiment 19 is the transgenic animal of any one of embodiments 1 to 18, wherein the transgenic animal is a canine.4937-9365-5942.1 45Atorney Docket No.: 069296.11209 / 28WO1

[0188] Embodiment 20 is the transgenic animal of embodiment 19, wherein the canine is selected from a short-eared dog (Atelocynus microtis), an African wolf (Canis lupaster), a coyote (Cants latrans), a dog (Canis familiaris), an Ethiopian wolf (Cants simensis), a golden jackal (Canis aureus), a gray wolf (Canis lupus), a crab-eating fox (Cerdocyon thous), a maned wolf (Chrysocyon brachyurus), a dhole (Ciion 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).

[0189] Embodiment 21 is a recombinant host cell comprising at least one direwolf (Aenocyon dints) gene variant associated with(a) body size, muscle mass, and / or metabolism;(b) craniofacial morphology; and / or(c) neurobehavior.

[0190] Embodiment 22 is the recombinant host cell of embodiment 21, wherein the at least one direwolf (Aenocyon dints) gene variant associated with body size, muscle mass, and / or metabolism is selected from the group consisting of an ADAMTS9 variant, a LCORL variant, a MSTN variant, a LCORL-NCAPG variant, an ADAM10 variant, an APOH variant, a BCKDHB variant, a BRAP variant, a CHCHD5 variant, a CHRM3 variant, a CPT1C variant, a DBN1 variant, an EPDR1 variant, an ETNPPL variant, a FABP4 variant, a HMGA2 variant, a KBTBD13 variant, a LDLR variant, a MAP2K5 variant, a NCOR1 variant, aNTRK3 variant, aPCKl variant, aPLEKHSl variant, aPNLIPRPl variant, a PPFIBP1 variant, a SLC30A8 variant, a ST3GAL2 variant, a STX16 variant, a WIFI variant, a WNT2 variant, a WWOX variant, and a ZFPM2 variant.

[0191] Embodiment 23 is the recombinant host cell of embodiment 21, wherein the at least one direwolf (Aenocyon dirus) gene variant associated with craniofacial morphology is selected from the group consisting of an ATXN10 variant, a CACNA1S variant, a CAPZB variant, a CDH24 variant, a DLX3 variant, a GAS6 variant, a GRK.1 variant, aHMGA2 variant, aMAPREl variant, aMAPRE2 variant, aMCHRl variant, aNBLl variant, an OSR1 variant, a POLR1 A variant, a PSMB11 variant, a PTCD3 variant, a SIK3 variant, a TMCC1 variant, aTMEM25B variant, a WIFI variant, aXPNPEP3 variant, and aZFP36Ll variant.4937-9365-5942.1 46Atorney Docket No.: 069296.11209 / 28WO1

[0192] Embodiment 24 is the recombinant host cell of embodiment 21, wherein the at least one direwolf (Aenocyon dirus) gene variant associated with neurobehavior is selected from a NXPH2 variant and a SLIT2 variant.

[0193] Embodiment 25 is the recombinant host cell of embodiment 22, wherein the ADAMTS9 variant, the LCORL variant, and / or the MSTN variant are loss-of-function variants.

[0194] Embodiment 26 is the recombinant host cell of any one of embodiments 21-25, wherein the direwolf gene variant comprises at least one change in the nucleotide sequence of the gene.

[0195] Embodiment 27 is the recombinant host cell of embodiment 26, wherein the change in the nucleotide sequence is a substitution, an insertion, a deletion, or a combination thereof.

[0196] Embodiment 28 is the recombinant host cell of embodiment 27, 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.

[0197] Embodiment 29 is the recombinant host cell of embodiment 27, wherein the substitution, the insertion, the deletion, or a combination thereof is in a regulatory region of the gene.

[0198] Embodiment 30 is the recombinant host cell of embodiment 22, wherein the direwolf gene variant associated with body size, muscle mass, and / or metabolism 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: 1-32 and combinations thereof.

[0199] Embodiment 31 is the recombinant host cell of embodiment 22, wherein the direw olf gene variant associated with body size, muscle mass, and / or metabolism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:70-101, and combinations thereof.

[0200] Embodiment 32 is the recombinant host cell of embodiment 23, wherein the direwolf gene variant associated with craniofacial morphology 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: 16, 29, 33-52, and combinations thereof.

[0201] Embodiment 33 is the recombinant host cell of embodiment 23, wherein the direw olf gene variant associated with craniofacial morphology comprises a nucleotide sequence4937-9365-5942.1 47Attorney Docket No.: 069296.11209 / 28WO1selected from the group consisting of SEQ ID NOs:85, 98, 102-121, and combinations thereof.

[0202] Embodiment 34 is the recombinant host cell of embodiment 24, wherein the direwolf gene variant associated with neurobehavior 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: 68-69 and combinations thereof.

[0203] Embodiment 35 is the recombinant host cell of embodiment 24, wherein the direwolf gene variant associated with neurobehavior comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 137-138 and combinations thereof.

[0204] Embodiment 36 is the recombinant host cell of any one of embodiments 21-35, wherein the recombinant host cell comprises at least 2, 3, 4. 5, 6, 7. 8, 9, 10, 15, 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) direwolf gene variants.

[0205] Embodiment 37 is the recombinant host cell of any one of embodiments 21 to 36, wherein the recombinant host cell fails to express an endogenous homologue of at least one of ADAMTS9. LCORL, MSTN, LCORL-NCAPG, ADAM 10, APOH, BCKDHB, BRAP, CHCHD5, CHRM3. CPT1C, DBN1, EPDR1. ETNPPL, FABP4. HMGA2. KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3. SIK3, TMCC1, TMEM25B. XPNPEP3. ZFP36L1, NXPH2, and SLIT2.

[0206] Embodiment 38 is the recombinant host cell of embodiment 37, wherein the recombinant host cell fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54 (or any number in between) of ADAMTS9, LCORL, MSTN. LCORL-NCAPG, ADAM10, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3. SIK3, TMCC1, TMEM25B. XPNPEP3. ZFP36L1, NXPH2, and SLIT2.

[0207] Embodiment 39 is the recombinant host cell of any one of embodiments 21 to 38, wherein the recombinant host cell is a canine cell.

[0208] Embodiment 40 is the recombinant host cell of embodiment 39, wherein the canine cell is selected from 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 wolf4937-9365-5942.1 48Atorney Docket No.: 069296.11209 / 28WO1(Cants simensis) cell, a golden jackal (Cants aureus) cell, a gray wolf Cants lupus) cell, a crab-eating fox (Cerdocyon thous) cell, a maned wolf (Chrysocyon brachyurus) cell, a dhole (Cuon alptnus) 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 grtseus) cell, or an African wild dog (Lycaon pictus) cell.

[0209] Embodiment 41 is the recombinant host cell of any one of embodiments 21 to 38, wherein the recombinant host cell is a stem cell.

[0210] Embodiment 42 is the recombinant host cell of claim 41. wherein the stem cell is selected from an induced pluripotent stem cell (iPSC), an embryonic stem (ES) cell, or a mesenchymal stem cell (MSC).

[0211] Embodiment 43 is the recombinant host cell of any one of embodiments 21 to 38, wherein the recombinant host cell is a reprogrammed cell.

[0212] Embodiment 44 is the recombinant host cell of any one of embodiments 21 to 38, wherein the recombinant host cell is a fibroblast cell or a mesenchymal cell.

[0213] Embodiment 45 is the recombinant host cell of any one of embodiments 21 to 38, wherein the recombinant host cell is an endothelial progenitor cell (EPC) or a pericyte.

[0214] Embodiment 46 is the recombinant host cell of any one of embodiments 21 to 38, 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.

[0215] Embodiment 47 is a method of creating a transgenic animal with a body size, muscle mass, and / or metabolism of a direwolf; a craniofacial morphology of a direwolf; and / or a neurobehavior 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 body¬ size, muscle mass, and / or metabolism; at least one direwolf gene variant associated with craniofacial morphology; and / or at least one direwolf gene variant associated with neurobehavior; 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 body size, muscle mass, and / or metabolism of a direwolf; the craniofacial morphology of a direwolf; and / or the neurobehavior of a direwolf.4937-9365-5942.1 49Atorney Docket No.: 069296.11209 / 28WO1

[0216] Embodiment 48 is the method of embodiment 47, wherein the at least one direwolf (Aenocyon dims) gene variant associated with body size, muscle mass, and / or metabolism is selected from the group consisting of an ADAMTS9 variant, a LCORL variant, a MSTN variant, a LCORL-NCAPG variant, an ADAMI 0 variant, an APOH variant, a BCKDHB variant, a BRAP variant, a CHCHD5 variant, a CHRM3 variant, a CPT1C variant, a DBN1 variant, an EPDR1 variant, an ETNPPL variant, a FABP4 variant, a HMGA2 variant, a KBTBD13 variant. aLDLR variant, aMAP2K5 variant, aNCORl variant. aNTRK3 variant, a PCK1 variant, a PLEKHS1 variant, a PNLIPRP1 variant, a PPFIBP1 variant, a SLC30A8 variant, a ST3GAL2 variant, a STX16 variant, a WIFI variant, a WNT2 variant, a WWOX variant, and a ZFPM2 variant.

[0217] Embodiment 49 is the method of embodiment 47, wherein the at least one direwolf (Aenocyon dims) gene variant associated with craniofacial morphology is selected from the group consisting of an ATXN10 variant, a CACNA1S variant, a CAPZB variant, a CDH24 variant, a DLX3 variant, a GAS6 variant, a GRK1 variant, a HMGA2 variant, a MAPRE1 variant, aMAPRE2 variant, aMCHRl variant, aNBLl variant, an OSR1 variant, aPOLRIA variant, a PSMB11 variant, a PTCD3 variant, a SIK.3 variant, a TMCC1 vanant, a TMEM25B variant, a WIFI variant, aXPNPEP3 variant, and aZFP36Ll variant.

[0218] Embodiment 50 is the method of embodiment 47, wherein the at least one direwolf (Aenocyon dims) gene variant associated with neurobehavior is selected from aNXPH2 variant and a SLIT2 variant.

[0219] Embodiment 51 is the method of embodiment 48, wherein the ADAMTS9 variant, the LCORL variant, the MSTN variant, and combinations thereof are loss-of-function variants.

[0220] Embodiment 52 is the method of embodiment 47, wherein the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene.

[0221] Embodiment 53 is the method of embodiment 52, wherein the change in the nucleotide sequence is a substitution, an insertion, a deletion, or a combination thereof.

[0222] Embodiment 54 is the method of embodiment 53, 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.

[0223] Embodiment 55 is the method of embodiment 53, wherein the substitution, the insertion, the deletion, or a combination thereof is in a regulatory region of the gene.4937-9365-5942.1 50Atorney Docket No.: 069296.11209 / 28WO1

[0224] Embodiment 56 is the method of embodiment 48, wherein the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism 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: 1-32 and combinations thereof.

[0225] Embodiment 57 is the method of embodiment 48, wherein the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:70-101, and combinations thereof.

[0226] Embodiment 58 is the method of embodiment 49, wherein the at least one direwolf gene variant associated with craniofacial morphology 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: 16, 29, 33-52, and combinations thereof.

[0227] Embodiment 59 is the method of embodiment 49, wherein the at least one direwolf gene variant associated with craniofacial morphology comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:85, 98, 102-121, and combinations thereof.

[0228] Embodiment 60 is the method of embodiment 50, wherein the at least one direwolf gene variant associated with neurobehavior 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: 68-69 and combinations thereof.

[0229] Embodiment 61 is the method of embodiment 50, wherein the at least one direwolf gene variant associated with neurobehavior comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 137-138 and combinations thereof.

[0230] Embodiment 62 is the method of embodiment 47, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) direwolf gene variants are introduced into the cell.

[0231] Embodiment 63 is the method of any one of embodiments 47 to 62, wherein the transgenic animal fails to express an endogenous homologue of at least one of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAMI 0, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1. PLEKHS1, PNLIPRP1, PPFIBP1. SLC30A8.ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB,4937-9365-5942.1 51Atorney Docket No.: 069296.11209 / 28WO1CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, 0SR1, POLR1A. PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3. ZFP36L1, NXPH2, and SLIT2.

[0232] Embodiment 64 is the method of embodiment 63, wherein the transgenic animal fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54 (or any number in between) of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAM10, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13. LDLR. MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2. and SLIT2.

[0233] Embodiment 65 is the method of any one of embodiments 47-64, wherein the cell is a stem cell.

[0234] Embodiment 66 is the method of embodiment 65, wherein the stem cell is selected from an induced stem cell, an embryonic stem (ES) cell, or a mesenchymal stem cell (MSC).

[0235] Embodiment 67 is the method of any one of embodiments 47-64, wherein the cell is a reprogrammed cell.

[0236] Embodiment 68 is the method of any one of embodiments 47-64, wherein the cell is a fibroblast cell or a mesenchymal cell.

[0237] Embodiment 69 is the method of any one of embodiments 47-64, 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.

[0238] Embodiment 70 is the method of any one of embodiments 47-64, wherein the cell is an endothelial progenitor cell (EPC) or a pericyte.

[0239] Embodiment 71 is the method of any one of embodiments 47-64, wherein the animal is a canine.

[0240] Embodiment 72 is the method of embodiment 71, wherein the canine is selected from a short-eared dog (Atelocynus microtis), an African wolf (Canis lupaste ), 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 hrachyurus), 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 (Lycalopex4937-9365-5942.1 52Attorney Docket No.: 069296.11209 / 28WO1gymnocercus), a Sechuran fox (Lycalopex sechurae), a South American gray fox Lycalopex griseus). or an African wild dog (Lycaon pic / us).

[0241] Embodiment 73 is a transgenic animal made by the method of any one of embodiments 47-72.

[0242] 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

[0243] Example 1: Creation of Transgenic Animals comprising direwolf gene variants.

[0244] 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.

[0245] Materials and Methods:

[0246] Cell Engineering:

[0247] 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 aHISTOPAQUE® density7gradient. 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.

[0248] Genome Engineering:

[0249] As a proof of concept, five loci or genes of interest from Table 1 that are associated with core direwolf phenoty pes were targeted. The genes are: LCORL, FGF5, MC1R, MFSD12, and MSTN. 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 gene4937-9365-5942.1 53Atorney Docket No.: 069296.11209 / 28WO1knock-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.

[0250] Somatic Cell Nuclear Transfer:

[0251] 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).

[0252] Analysis:

[0253] 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).

[0254] 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.

[0255] 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, ET 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.4937-9365-5942.1 54Atorney Docket No.: 069296.11209 / 28WO1

[0256] 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.

[0257] Birth and development of gene-edited direwolves

[0258] On October 1. 2024, two male Colossal Dire Wolves were bom, 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.

[0259] 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 subadult 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.

[0260] 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.4937-9365-5942.1 55

Claims

1. Atorney Docket No.: 069296.11209 / 28WO1CLAIMSIt is claimed:

1. A transgenic animal comprising at least one direwolf (Aenocyon dims) gene variant associated with(a) body size, muscle mass, and / or metabolism;(b) craniofacial morphology; and / or(c) neurobehavior.

2. The transgenic animal of claim 1, wherein the at least one direwolf (Aenocyon dirus) gene variant associated with body size, muscle mass, and / or metabolism is selected from the group consisting of an ADAM metallopeptidase with thrombospondin type 1 motif 9 (ADAMTS9) variant, a Ligand dependent nuclear receptor corepressor like (LCORL) variant, a Myostatin (MSTN) variant, a Ligand dependent nuclear receptor corepressor like and chromosome condensation protein G (LCORL-NCAPG) variant, an A disintegrm and metallopeptidase domain 10 (ADAM10) variant, an Apolipoprotein H (APOH) variant, a Branched chain keto acid dehydrogenase El subunit beta (BCKDHB) variant, a BRCA1 associated protein (BRAP) variant, a Coiled-coil-helix-coiled-coil-helix domain containing 5 (CHCHD5) variant, a Muscarinic acetylcholine receptor M3 (CHRM3) variant, a Carnitine palmitoyltransferase 1C (CPT1C) variant, a Drebrin 1 (DBN1) variant, an Emendymin related 1 (EPDR1) variant, an Ethanolamine-phosphate phospholyase (ETNPPL) variant, a Fatty acid binding protein 4 (FABP4) variant, a High mobility' group AT-hook 2 (HMGA2) variant, a Kelch repeat and BTB domain containing 13 (KBTBD13) variant, a Low-density lipoprotein receptor (LDLR) variant, a Mitogen-activated protein kinase kinase 5 (MAP2K5) variant, a Nuclear receptor corepressor 1 (NCORl) variant, a Neurotrophic receptor tyrosine kinase 3 (NTRK3) variant, a Phosphoenolpyruvate carboxykinase 1 (PCK1) variant, a Pleckstrin homology domain containing SI (PLEKHS1) variant, a Pancreatic lipase related protein 1 (PNLIPRP1) variant. aPPFIA binding protein 1 (PPFIBP1) variant, a Solute earner family 30 member 8 (SLC30A8) variant, a ST3 beta-galactoside-alpha-2, 3-sialytransferase 2 (ST3GAL2) variant, a Syntaxin 16 (STX16) variant, a WNT inhibitory factor 1 (WIFI) variant, a Wingless ty pe MMTV integration site family member 2 (WNT2) variant, a WW domain containing oxidoreductase (WWOX) variant, and aZinc finger protein FOG family member 2 (ZFPM2) variant.

3. The transgenic animal of claim 1, wherein the at least one direwolf (Aenocyon dims) gene variant associated with craniofacial morphology is selected from the group consisting of4937-9365-5942 1Atorney Docket No.: 069296.11209 / 28WO1an Ataxin 10 (ATXN10) variant, a Calcium voltage-gated channel subunit alpha IS (CACNA1S) variant, a Capping actin protein of muscle Z-line subunit beta (CAPZB) variant, a Cadherin 24 (CDH24) variant, a Distal-less homeobox 3 (DLX3) variant, a Growth arrest specific 6 (GAS6) variant, a G protein coupled receptor kinase 1 (GRK1) variant, a High mobility7group AT-hook 2 (HMGA2) variant, a Microtubule associated protein RP / EB family member 1 (MAPRE1) variant, a Microtubule associated protein RP / EB family member 2 (MAPRE2) variant, a Melanin concentrating hormone receptor 1 (MCHR1) variant, a DAN family BMP antagonist (NBL1) variant, an Odd-skipped related transcription factor 1 (OSR1) variant, a RNA polymerase I subunit A (POLR1 A) variant, a Proteasome subunit beta 11 (PSMB11) variant, a Pentatricopeptide repeat domain 3 (PTCD3) variant, a SIK family kinase 3 (SIK3) variant, a Transmembrane and coiled-coil domain family 1 (TMCC1) variant, a Transmembrane protein 255B (TMEM25B) variant, a X-prolyl aminopeptidase 3 (XPNPEP3) variant, a WNT inhibitory factor 1 (WIFI) variant, and aZFP36 ring finger protein like 1 (ZFP36L1) variant.

4. The transgenic animal of claim 1, wherein the at least one direwolf (Aenocyon dirus) gene variant associated with neurobehavior is selected from a Neurexophilin 2 (NXPH2) variant and a Slit guidance ligand 1 (SLIT2) variant.

5. The transgenic animal of claim 2, wherein the ADAMTS9 variant, the LCORL variant, the MSTN variant, and combinations thereof are loss-of-function variants.

6. 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.

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

8. The transgenic animal of claim 7, 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.

9. The transgenic animal of claim 7, wherein the substitution, the insertion, the deletion, or a combination thereof is in a regulatory region of the gene.

10. The transgenic animal of claim 2, wherein the at least one direwolf gene vanant associated with body size, muscle mass, and / or metabolism comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity7to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-32 and combinations thereof.4937-9365-5942.1 57Atorney Docket No.: 069296.11209 / 28WO111. The transgenic animal of claim 2, wherein the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:70-101, and combinations thereof.

12. The transgenic animal of claim 3, wherein the at least one direwolf gene variant associated with craniofacial morphology 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: 16, 29, 33-52, and combinations thereof.

13. The transgenic animal of claim 3, wherein the at least one direwolf gene variant associated with craniofacial morphology' comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:85, 98, 102-121, and combinations thereof.

14. The transgenic animal of claim 4, wherein the at least one direwolf gene variant associated with neurobehavior 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: 68-69 and combinations thereof.

15. The transgenic animal of claim 4, wherein the at least one direwolf gene variant associated with neurobehavior comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 137-138 and combinations thereof.

16. The transgenic animal of claim 1, wherein the transgenic animal comprises at least 2, 3, 4, 5, 6. 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) direwolf gene variants.

17. The transgenic animal of any one of claims 1 to 16, wherein the transgenic animal fails to express an endogenous homologue of at least one of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAM10, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13. LDLR. MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2. and SLIT2.

18. The transgenic animal of claim 17. wherein the transgenic animal fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54 (or any number in between) of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAMI 0, APOH, BCKDHB, BRAP, CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2. KBTBD13, LDLR, MAP2K5. NCORL NTRK3, PCK1, PLEKHS1, PNLIPRPL PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX,4937-9365-5942.1 58Atorney Docket No.: 069296.11209 / 28WO1ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2.

19. The transgenic animal of any one of claims 1 to 18, wherein the transgenic animal is a canine.

20. The transgenic animal of claim 19. 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 j ackal (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).

21. A recombinant host cell comprising at least one direwolf (Aenocyon dirus) gene variant associated with(d) body size, muscle mass, and / or metabolism;(e) craniofacial morphology: and / or(1) neurobehavior.

22. The recombinant host cell of claim 21 , wherein the at least one direwolf (Aenocyon dirus) gene variant associated with body size, muscle mass, and / or metabolism is selected from the group consisting of an ADAMTS9 variant, a LCORL variant, a MSTN variant, a LCORL-NCAPG variant, an ADAM 10 variant, an APOH variant, a BCKDHB variant, a BRAP variant, a CHCHD5 variant, a CHRM3 variant, a CPT1C variant, a DBN1 variant, an EPDR1 variant, an ETNPPL variant, aFABP4 variant, aHMGA2 variant, aKBTBD13 variant, a LDLR variant, aMAP2K5 variant, aNCORl variant, aNTRK3 variant, aPCKl variant, aPLEKHSl variant. aPNLIPRPl variant, aPPFIBPl variant, a SLC30A8 variant, a ST3GAL2 variant, a STX16 variant, a WIFI variant, a WNT2 variant, a WWOX variant, and a ZFPM2 variant.

23. The recombinant host cell of claim 21, wherein the at least one direwolf (Aenocyon dirus) gene variant associated with craniofacial morphology' is selected from the group consisting of an ATXN10 variant, a CACNA1S variant, a CAPZB variant, a CDH24 variant, a DLX3 variant, a GAS6 variant, a GRK1 variant, a HMGA2 variant, a MAP RE 1 variant, a4937-9365-5942.1 59Atorney Docket No.: 069296.11209 / 28WO1MAPRE2 variant, a MCHR1 variant, a NBL1 variant, an OSR1 variant, a POLR1 A variant, a PSMB11 variant, a PTCD3 variant, a SIK3 variant, a TMCC1 variant, a TMEM25B variant. aXPNPEP3 variant, a WIFI variant, and aZFP36Ll variant.

24. The recombinant host cell of claim 21, wherein the at least one direwolf (Aenocyon dims) gene variant associated with neurobehavior is selected from a NXPH2 variant and a SLIT2 variant.

25. The recombinant host cell of claim 22, wherein the ADAMTS9 variant, the LCORL variant, the MSTN variant, and combinations thereof are loss-of-function variants.

26. The recombinant host cell of claim 21, wherein the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene.

27. The recombinant host cell of claim 26. wherein the change in the nucleotide sequence is a substitution, an insertion, a deletion, or a combination thereof.

28. The recombinant host cell of claim 27, 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.

29. The recombinant host cell of claim 27, wherein the substitution, the insertion, the deletion, or a combination thereof is in a regulatory region of the gene.

30. The recombinant host cell of claim 22, wherein the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism 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: 1-32 and combinations thereof.

31. The recombinant host cell of claim 22, wherein the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 70- 101, and combinations thereof.

32. The recombinant host cell of claim 23, wherein the at least one direwolf gene variant associated with craniofacial morphology 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:

16. 29, 33-52, and combinations thereof.

33. The recombinant host cell of claim 23, wherein the at least one direwolf gene variant associated with craniofacial morphology comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 85, 98, 102-121, and combinations thereof.

34. The recombinant host cell of claim 24, wherein the at least one direwolf gene variant associated with neurobehavior comprises a nucleotide sequence having at least 80%. at least4937-9365-5942.1 60Atorney Docket No.: 069296.11209 / 28WO185%, at least 90%, or at least 95% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 68-69 and combinations thereof.

35. The recombinant host cell of claim 24, wherein the at least one direwolf gene variant associated with neurobehavior comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 137-138 and combinations thereof.

36. The recombinant host cell of claim 21. wherein the recombinant host cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) direwolf gene variants.

37. The recombinant host cell of any one of claims 21 to 36, wherein the recombinant host cell fails to express an endogenous homologue of at least one of ADAMTS9, LCORL, MSTN, LCORL-NCAPG, ADAM10, APOH, BCKDHB. BRAP. CHCHD5, CHRM3, CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11. PTCD3, SIK3. TMCC1. TMEM25B, XPNPEP3, ZFP36L1, NXPH2. and SLIT2.

38. The recombinant host cell of claim 37, wherein the recombinant host cell fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54 (or any number in between) of ADAMTS9, LCORL, MSTN, LCORL-NCAPG. ADAM10, APOH. BCKDHB. BRAP. CHCHD5, CHRM3. CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2.

39. The recombinant host cell of any one of claims 21 to 38, wherein the recombinant host cell is a canine cell.

40. The recombinant host cell of claim 39. wherein the canine cell is selected from a short-eared dog (Atelocynus microtis) cell, an African wolf Canis iupaster) cell, a coyote Canis latrans) cell, a dog (Canis familiaris) cell, an Ethiopian wol f (Canis simensis) cell, a golden jackal Canis aureus) cell, a gray wolf Canis lupus) cell, a crab-eating fox (Cerdocyon thous) cell, a maned w ol f (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 j ackal (Lupulella mesomeias) cell, a side-striped jackal (Lupulella4937-9365-5942.1 61Atorney Docket No.: 069296.11209 / 28WO1aduslu ) 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.

41. The recombinant host cell of any one of claims 21 to 38, wherein the recombinant host cell is a stem cell.

42. The recombinant host cell of claim 41, wherein the stem cell is selected from an induced pluripotent stem cell (iPSC), an embryonic stem (ES) cell, or a mesenchymal stem cell (MSC).

43. The recombinant host cell of any one of claims 21 to 38, wherein the recombinant host cell is a reprogrammed cell.

44. The recombinant host cell of any one of claims 21 to 38, wherein the recombinant host cell is a fibroblast cell or a mesenchymal cell.

45. The recombinant host cell of any one of claims 21 to 38, wherein the recombinant host cell is an endothelial progenitor cell (EPC) or a pericyte.

46. The recombinant host cell of any one of claims 21 to 38, 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.

47. A method of creating a transgenic animal with a body size, muscle mass, and / or metabolism of a direwolf; a craniofacial morphology of a direwolf; and / or a neurobehavior 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 body size, muscle mass, and / or metabolism; at least one direwolf gene variant associated with craniofacial morphology; and / or at least one direwolf gene variant associated with neurobehavior; 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 body size, muscle mass, and / or metabolism of a direwolf; the craniofacial morphology of a direwolf; and / or the neurobehavior of a direwolf.

48. The method of claim 47, wherein the at least one direwolf (Aenocyon dims) gene variant associated with body size, muscle mass, and / or metabolism is selected from the group consisting of an ADAMTS9 variant, a LCORL variant, a MSTN variant, a LCORL-NCAPG variant, an ADAM10 variant, an APOH variant, a BCKDHB variant, a BRAP variant, a4937-9365-5942.1 62Atorney Docket No.: 069296.11209 / 28WO1CHCHD5 variant, a CHRM3 variant, a CPT1C variant, aDBNl variant, an EPDR1 variant, an ETNPPL variant, a FABP4 variant, a HMGA2 variant, a KBTBD13 variant, a LDLR variant, a MAP2K5 variant, aNCORl variant, aNTRK.3 variant, a PCK1 variant, a PLEKHS1 variant, aPNLIPRPl variant, a PPFIBP1 variant, a SLC30A8 variant, a ST3GAL2 variant, a STX16 variant, a WIFI variant, a WNT2 variant, a WWOX variant, and a ZFPM2 variant.

49. The method of claim 47, wherein the at least one direwolf (Aenocyon dims) gene variant associated with craniofacial morphology is selected from the group consisting of an ATXN10 variant, a CACNA1S variant, a CAPZB variant, a CDH24 variant, a DLX3 variant, aGAS6 variant, a GRK1 variant, aHMGA2 variant, aMAPREl variant, aMAPRE2 variant, a MCHR1 variant, a NBL1 variant, an OSR1 variant, a POLR1 A variant, a PSMB11 variant, aPTCD3 variant, a SIK3 variant, a TMCCl variant, aTMEM25B variant, a WIFI variant, a XPNPEP3 variant, and aZFP36Ll variant.

50. The method of claim 47, wherein the at least one direwolf (Aenocyon dims) gene variant associated with neurobehavior is selected from aNXPH2 variant and a SLIT2 variant.

51. The method of claim 48, wherein the ADAMTS9 variant, the LCORL variant, the MSTN variant, and combinations thereof are loss-of-function variants.

52. The method of claim 47, wherein the at least one direwolf gene variant comprises at least one change in the nucleotide sequence of the gene.

53. The method of claim 52. wherein the change in the nucleotide sequence is a substitution, an insertion, a deletion, or a combination thereof.

54. The method of claim 53, 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.

55. The method of claim 53, wherein the substitution, the insertion, the deletion, or a combination thereof is in a regulatory region of the gene.

56. The method of claim 48, wherein the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism 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: 1-32 and combinations thereof.

57. The method of claim 48, wherein the at least one direwolf gene variant associated with body size, muscle mass, and / or metabolism comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:70-101, and combinations thereof.4937-9365-5942.1 63Atorney Docket No.: 069296.11209 / 28WO158. The method of claim 49, wherein the at least one direwolf gene variant associated with craniofacial morphology 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: 16, 29, 33-52, and combinations thereof.

59. The method of claim 49, wherein the at least one direwolf gene variant associated with craniofacial morphology comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:85, 98, 102-121, and combinations thereof.

60. The method of claim 50, wherein the at least one direwolf gene variant associated with neurobehavior 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: 68-69 and combinations thereof.

61. The method of claim 50, wherein the at least one direwolf gene variant associated with neurobehavior comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 137-138 and combinations thereof.

62. The method of claim 47, wherein at least 2, 3, 4, 5, 6, 7. 8, 9, 10, 15, 20, 25, 30, 40, 50, 51, 52, 53, 54 (or any number in between) direwolf gene variants are introduced into the cell.

63. The method of any one of claims 47 to 62, wherein the transgenic animal fails to express an endogenous homologue of at least one of ADAMTS9, LCORL, MSTN, LCORL-NCAPG. ADAM10, APOH. BCKDHB. BRAP. CHCHD5, CHRM3. CPT1C, DBN1, EPDR1, ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10, CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A, PSMB11, PTCD3, SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2.

64. The method of claim 63, wherein the transgenic animal fails to express the endogenous homologue of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54 (or any number in between) of ADAMTS9, LCORL, MSTN, LCORL-NCAPG.ADAM10. APOH. BCKDHB. BRAP. CHCHD5. CHRM3. CPT1C, DBN1, EPDR1.ETNPPL, FABP4, HMGA2, KBTBD13, LDLR, MAP2K5, NCOR1, NTRK3, PCK1, PLEKHS1, PNLIPRP1, PPFIBP1, SLC30A8, ST3GAL2, STX16, WIFI, WNT2, WWOX, ZFPM2, ATXN10. CACNA1S, CAPZB, CDH24, DLX3, GAS6, GRK1, MAPRE1, MAPRE2, MCHR1, NBL1, OSR1, POLR1A. PSMB11, PTCD3. SIK3, TMCC1, TMEM25B, XPNPEP3, ZFP36L1, NXPH2, and SLIT2.4937-9365-5942.1 64Atorney Docket No.: 069296.11209 / 28WO165. The method of any one of claims 47-64, wherein the cell is a stem cell.

66. The method of claim 65, wherein the stem cell is selected from an induced stem cell, an embryonic stem (ES) cell, or a mesenchymal stem cell (MSC).

67. The method of any one of claims 47-64, wherein the cell is a reprogrammed cell.

68. The method of any one of claims 47-64, wherein the cell is a fibroblast cell or a mesenchymal cell.

69. The method of any one of claims 47-64, 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.

70. The method of any one of claims 47-64, wherein the cell is an endothelial progenitor cell (EPC) or a pericyte.

71. The method of any one of claims 47-64, wherein the animal is a canine.

72. The method of claim 71, wherein the canine is selected from a short-eared dog (Atelocynus microtis), an African wolf (Canis lupaster), a coyote (Canis latrans), a dog (Canis familiar is), 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 j ackal (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 gr is eus), or an African wild dog (Lycaon pictus).

73. A transgenic animal made by the method of any one of claims 47-72.4937-9365-5942.1 65

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