Multiplex gene editing

Abstract

Materials and methods for making multiplex gene edits in cells and 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 - 24 . (canceled) 
     
     
         25 . A method of introducing multiple gene edits in a cell or embryo comprising simultaneously introducing into the cell
 a first targeted endonuclease directed to a first target chromosomal DNA site and a first homology directed repair (HDR) template that has homology to the first target chromosomal DNA site; and   a second targeted endonuclease directed to a second target chromosomal DNA site and a second HDR template that has homology to the second target chromosomal DNA site   wherein the first HDR template replaces the first target chromosomal DNA site and the second HDR template replaces the second target chromosomal DNA site.   
     
     
         26 . The method of  claim 25 , wherein the cell is a primary livestock cell or the embryo is a livestock embryo. 
     
     
         27 . The method of  claim 26 , wherein the livestock embryo is selected from the group consisting of a zygote, a blastocyst, morula, and 1-200 cells. 
     
     
         28 . The method of  claim 25 , further comprising simultaneously introducing into the cell one or more of:
 a third targeted endonuclease directed to a third chromosomal DNA site and a third HDR template that has homology to the third target chromosomal DNA site wherein the third HDR template replaces the third target chromosomal DNA site;   a fourth targeted endonuclease directed to a fourth chromosomal DNA site and a fourth HDR template that has homology to the fourth target chromosomal DNA site wherein the fourth HDR template replaces the fourth target chromosomal DNA site;   a fifth targeted endonuclease directed to a fifth chromosomal DNA site and a fifth HDR template that has homology to the fifth target chromosomal DNA site wherein the fifth HDR template replaces the fifth target chromosomal DNA site;   a sixth targeted endonuclease directed to a sixth chromosomal DNA site and a sixth HDR template that has homology to the sixth target chromosomal DNA site wherein the sixth HDR template replaces the sixth target chromosomal DNA site; and   a seventh targeted endonuclease directed to a seventh chromosomal DNA site and a seventh HDR template that has homology to the seventh target chromosomal DNA site wherein the seventh HDR template replaces the seventh target chromosomal DNA site.   
     
     
         29 . The method of  claim 28 , wherein the endonuclease is selected from transcription activator-like effector nuclease (TALEN) or CRISPR/cas9 and combinations thereof. 
     
     
         30 . The method of  claim 25 , wherein the first HDR template encodes a knockout of the first target chromosomal DNA site. 
     
     
         31 . The method of  claim 25 , wherein the first HDR template encodes an exogenous allele for replacement of an allele at the first target chromosomal DNA site. 
     
     
         32 . The method of  claim 29 , wherein the TALEN or CRISPR/cas9 is administered to the cell as mRNA. 
     
     
         33 . The method of  claim 29 , wherein the TALEN or CRISPR/cas9 is administered to the cell as a protein. 
     
     
         34 . The method of  claim 25 , wherein the first targeted endonuclease, the first HDR template, the second targeted endonuclease, and the second HDR template are introduced into the cell by electroporation, recombinant viruses, liposomes, microinjection, or calcium phosphate precipitation. 
     
     
         35 . The method of  claim 25 , wherein the first target chromosomal DNA site comprises a site in RAG2 and the second target chromosomal DNA site comprises a site in IL2Rγ. 
     
     
         36 . The method of  claim 28 , wherein the first target chromosomal DNA site comprises a site in RAG2, the second target chromosomal DNA site comprises a site in IL2Rγ, the third target chromosomal DNA site comprises a site in p53, the fourth target chromosomal DNA site comprises a site in APC, and the fifth target chromosomal DNA site comprises a site in LDLR. 
     
     
         37 . The method of  claim 28 , wherein the first target chromosomal DNA site comprises a site in NKX-2, the second target chromosomal DNA site comprises a site in GATA-4, and the third target chromosomal DNA site comprises a site in MESP1.