Multiplex gene editing

Abstract

Materials and methods for making multiplex gene edits in cells or embryos are presented. Further methods include animals and methods of making the same. Methods of making chimeric animals are presented, as well as chimeric animals.

1 . A large vertebrate animal comprising multiplex gene edits. 
     
     
         2 . The animal of  claim 1  comprising a number of knockout gene edits from 2-25. 
     
     
         3 . The animal of  claim 1  having a number of allele replacements from 2-25. 
     
     
         4 . The animal of  claim 3  having at least three allele replacements wherein each of the three alleles are for three different genes. 
     
     
         5 . The animal of  claim 3  comprising at least five exogenous replacement alleles. 
     
     
         6 . The animal of  claim 1  comprising a plurality of homozygous edits. 
     
     
         7 . The animal of  claim 1  being a chimera of host cells from a host animal and donor cells from a donor animal, with the host cells comprising the multiplex gene edits. 
     
     
         8 . The animal of  claim 7  wherein sexual reproduction of the animal transmits gametes having a genotype of the donor cells to progeny. 
     
     
         9 . The animal of  claim 1  being free of marker genes. 
     
     
         10 . The animal of  claim 1  being selected from the group consisting of livestock, simian, dog, cat, avian, bird, fish, rabbit, pig, cattle, buffalo, goat, sheep, and artiodactyl. 
     
     
         11 . The animal of  claim 1  being a first breed of cattle or a first breed of pig that has at least three native alleles replaced with corresponding exogenous alleles of a second breed or another species, wherein the animal is free of exogenous marker genes. 
     
     
         12 . The animal of  claim 1  wherein the multiplex gene edits are made in one or more of the following genes: IL2Rg, RAG2, DGAT, ABCG2, ACAN, AMELY, BLG, BMP 1B (FecB), DAZL, DGAT, Eif4GI, GDF8, Horn-poll locus, IGF2, CWC15, KissR/GRP54, OFD1Y, p65, PRLR, Prmd14, PRNP, Rosa, Socs2, SRY, ZFY, β-lactoglobulin, CLPG, MODY 1 (HNF4α), MODY 2 (GCK), MODY 3 (HNF1α), MODY 4 (Pdxl), MODY 5 (HNF-1β), MODY 6 (eurogenic differentiation 1), MODY 7 (KLF11), MODY 8 (CEL), MODY 9 (PAX4), MODY 10 (INS), MODY 11 (BLK), APC, ApoE, DMD, GHRHR, HR, HSD11B2, LDLR, NF1, NPPA, NR3C2, p53, PKD1, Rbm20, SCNN1G, tP53, FAH, HBB, IL2RG, PDX1, PITX3, Runx1, GGTA, VASA, MIWI, PIWI, DCAF17, VDR, PNPLA1, HRAS, Telomerase-vert, DSP, SNRPE, RPL21, LAMA3, UROD, EDAR, OFD1, PEX7, COL3A1, ALOX12B, HLCS, NIPAL4, CERS3, ANTXR1, B3GALT6, DSG4, UBR1, CTC1, MBTPS2, UROS, ABHD5, NOP10, ALMS1, LAMB3, EOGT, SAT1, RBPJ, ARHGAP31, ACVR1, IKBKG, LPAR6, HR, ATR, HTRA1, AIRE, BCS1L, MCCC2, DKC1, PORCN, EBP, SLITRK1, BTK, DOCK6, APCDD1, ZIP4, CASR, TERT, EDARADD, ATP6VOA2, PVRL1, MGP, KRT85, RAG-1, ROR2, CLAUDIN1, ABCAl2, SLA-DRA1, B4GALT7, COL7A1, NHP2, GNA11, WNT5A, USB1, LMNA, EPS8L3, NSDHL, TRPV3, KRAS, TINF2, TGM1, DCLRE1C, PKP1, WRAP53, KDM5C, ECM1, TP63, KRT14, RIPK4, PRKDC,BCL11a, BMI1, CCR5, CXCR4, DKK1, ETV2, FLI1, FLK1, GATA2, GATA4, HHEX, KIT, LMX1A, MYF5, MYOD1, MYOG, NKX2-5, NR4A2, PAX3, PDX1, PITX3, Runx1, RAG2, GGTA, HR, HANDII, TBX5, ETV2, PDX1, TBX4 ,ID2 SOX2, TTF1/NKX2-1, MESP1, GATA4, NKX2-5, FAH, PRKDC, RUNX1, FLI1, PITX3, LMX1A, DKK1, NR4A2/NURR1, FLK1, HHEX1, BCL11A, RAG2, RAG1, IL2RG, c-KIT/SCFR, BMI1, HANDII, TBX5, GATA2, OLIG1, OLIG2, heterozygotes thereof, homozygotes therefore, and combinations thereof. 
     
     
         13 . A method of making genetic edits in vitro in a vertebrate cell or embryo at a plurality of target chromosomal DNA sites comprising
 introducing into a vertebrate cell or embryo:
 a first targeted endonuclease directed to a first target chromosomal DNA site and a first homology directed repair (HDR) template homologous to the first target site sequence; and 
 a second targeted endonuclease directed to a second target chromosomal DNA site and a second HDR template homologous to the second target site sequence, 
 with the first HDR template sequence replacing the native chromosomal DNA sequence at the first target site and the second HDR template sequence replacing the native chromosomal DNA sequence at the second target site sequence. 
   
     
     
         14 . The method of  claim 13  further comprising
 introducing into the cell or embryo one or more of: 
 a third, fourth, fifth, sixth, and seventh targeted endonuclease directed to a third, fourth, fifth, sixth, and seventh target chromosomal DNA site, respectively, and 
 a third, fourth, fifth, sixth, and seventh HDR template homologous to the third, fourth, fifth, sixth, and seventh target chromosomal DNA sites, respectively. 
 
     
     
         15 . The method of  claim 13  wherein the targeted endonuclease comprises a TALENs, a zinc finger nuclease, RNA-guided endonuclease, a Cas9, or a combination thereof. 
     
     
         16 . The method of  claim 13  wherein at least the first target chromosomal DNA site is a locus for an allele. 
     
     
         17 . The method of  claim 16  wherein at least one of said HDR templates encodes a knockout of the DNA target site allele that is cognate to the template or at least one of said HDR templates encodes an exogenous allele for replacement of an allele at the DNA target site cognate to the template. 
     
     
         18 . The method of  claim 16  with the cell or embryo being a first species or a first breed of livestock, and a plurality of the edits comprise a replacement of a native allele with an exogenous allele from a second species or a second breed of animal. 
     
     
         19 . The method of  claim 16  wherein the cell or embryo is homozygous for a plurality of gene edits at the target DNA sites, said edits being encoded by HDR templates cognate to said target DNA sites. 
     
     
         20 . The method of  claim 16  with the cell or embryo being selected from the group consisting of large vertebrate, livestock, simian, dog, cat, avian, bird, fish, rabbit, pig, cattle, buffalo, goat, sheep, and artiodactyl, or with th the cell being selected from the group consisting of zygote, stem cell, adult stem cell, pluripotent cell, progenitor cell, and primary cell, or with the embryo being zygote, blastocyst, morula, or having a number of cells from 1-200. 
     
     
         21 . The method of  claim 16  wherein one or more of the target site sequences are chosen from the following list: IL2Rg, RAG2, DGAT, ABCG2, ACAN, AMELY, BLG, BMP 1B (FecB), DAZL, DGAT, Eif4GI, GDF8, Horn-poll locus, IGF2, CWC15, KissR/GRP54, OFD1Y, p65, PRLR, Prmd14, PRNP, Rosa, Socs2, SRY, ZFY, β-lactoglobulin, CLPG, MODY 1 (HNF4α), MODY 2 (GCK), MODY 3 (HNF1α), MODY 4 (Pdxl), MODY 5 (HNF-1β), MODY 6 (eurogenic differentiation 1), MODY 7 (KLF11), MODY 8 (CEL), MODY 9 (PAX4), MODY 10 (INS), MODY 11 (BLK), APC, ApoE, DMD, GHRHR, HR, HSD11B2, LDLR, NF1, NPPA, NR3C2, p53, PKD1, Rbm20, SCNN1G, tP53, FAH, HBB, IL2RG, PDX1, PITX3, Runx1, RAG2, GGTA, VASA, MIWI, PIWI, DCAF17, VDR, PNPLA1, HRAS, Telomerase-vert, DSP, SNRPE, RPL21, LAMA3, UROD, EDAR, OFD1, PEX7, COL3A1, ALOX12B, HLCS, NIPAL4, CERS3, ANTXR1, B3GALT6, DSG4, UBR1, CTC1, MBTPS2 ,UROS, ABHD5, NOP10, ALMS1, LAMB3, EOGT, SAT1, RBPJ, ARHGAP31, ACVR1, IKBKG, LPAR6, HR, ATR, HTRA1, AIRE, BCS1L, MCCC2, DKC1, PORCN, EBP, SLITRK1, BTK, DOCK6, APCDD1, ZIP4, CASR, TERT, EDARADD, ATP6V0A2, PVRL1, MGP, KRT85, RAG2, RAG-1, ROR2, CLAUDIN1, ABCAl2, SLA-DRA1, B4GALT7, COL7A1, NHP2, GNA11, WNT5A, USB1, LMNA, EPS8L3, NSDHL, TRPV3, KRAS, TINF2, TGM1, DCLRE1C, PKP1, WRAP53, KDM5C, ECM1, TP63, KRT14, RIPK4, PRKDC, BCL11a, BMI1, CCR5, CXCR4, DKK1, ETV2, FLI1, FLK1, GATA2, GATA4, HHEX, KIT, LMX1A, MYF5, MYOD1, MYOG, NKX2-5, NR4A2, PAX3, PDX1, PITX3, Runx1, GGTA, HR, HANDII, TBX5, ETV2, PDX1, TBX4, ID2 SOX2, TTF1/NKX2-1, MESP1, GATA4, NKX2-5, FAH, PRKDC, RUNX1, FLI1, PITX3, LMX1A, DKK1, NR4A2/NURR1, FLK1, HHEX1, BCL11A, RAG2, RAG1, IL2RG, c-KIT/SCFR, BMI1, HANDII, TBX5, GATA2, OLIG1, OLIG2, heterozygotes thereof, homozygotes therefore, and combinations thereof. 
     
     
         22 . A method of making multiplex gene knockouts in a primary vertebrate cell or embryo comprising introducing into the cell or embryo a plurality of TALENs targeted to different target genes in a presence of HDR templates with homology to said different target genes. 
     
     
         23 . A vertebrate embryo or vertebrate animal chimeric for host cells and donor cells comprising
 a host embryo with a plurality of host cell genetic edits at different chromosomal DNA sites, and   a donor cell integrated with the host cells to form the chimeric embryo or chimeric animal.   
     
     
         24 . A method of making a vertebrate embryo chimeric for host cells and donor cells comprising:
 introducing into a host vertebrate cell:
 a first targeted endonuclease directed to a first target chromosomal DNA site and a first homology directed repair (HDR) template homologous to the first target site sequence; and 
 a second targeted endonuclease directed to a second target chromosomal DNA site and a second HDR template homologous to the second target site sequence, 
   with the first HDR template sequence replacing the native chromosomal DNA sequence at the first target site and the second HDR template sequence replacing the native chromosomal DNA sequence at the second target site sequence,   cloning the cell to develop a host embryo, and   placing a donor cell in the host embryo.