Production of fmdv-resistant livestock by allele substitution

Abstract

A genetically modified livestock animal comprising a genomic modification to an eIF4G gene. Cells, genes, and proteins encompassing a protease-resistant eIF4G protein or gene.

What is claimed is: 
     
         1 . A gene edited livestock animal whose genome comprises a genome edited eIF4GI gene,
 wherein the genome edited eIF4GI gene expresses an altered eIF4GI protein relative to a wild type eIF4GI protein such that the gene edited livestock animal is resistant to cleavage by a proteinase of foot-and-mouth disease virus enzyme wherein the livestock animal is a cow or pig.   
     
     
         2 . The gene edited livestock animal of  claim 1 , wherein the genome edited eIF4GI gene comprises an insertion, a deletion, or a substitution of one or more bases of the eIF4GI gene. 
     
     
         3 . The gene livestock animal of  claim 1 , wherein the livestock animal is from a first breed and the genome edited eIF4GI gene comprises a natural allele of the eIF4GI gene found in another breed of the livestock animal. 
     
     
         4 . The gene edited livestock animal of  claim 1 , wherein the livestock animal is from a first species and the genome edited eIF4GI gene comprises an allele of the eIF4GI gene in a second species. 
     
     
         5 . The gene edited livestock animal of  claim 1 , wherein the livestock animal is homozygous for the genome edited eIF4GI gene. 
     
     
         6 . The gene edited livestock animal of  claim 1 , wherein the cow or pig is a founder animal. 
     
     
         7 . The gene edited livestock animal of  claim 1 , wherein the altered eIF4GI protein is edited to prevent binding of (i) a leader proteinase (L Pro ), (ii) a 3C protease (C Pro ), or (iii) L Pro  and C Pro  of foot-and-mouth disease virus enzyme. 
     
     
         8 . The gene edited livestock animal of  claim 1 , wherein the genome edited eIF4GI gene has the nucleotide sequence of SEQ ID NO: 2 and the livestock animal is the cow. 
     
     
         9 . The gene edited livestock animal of  claim 1 , wherein the genome edited eIF4GI gene has the nucleotide sequence of SEQ ID NO: 3. 
     
     
         10 . The gene edited livestock animal of  claim 1 , wherein the genome edited eIF4GI gene has the nucleotide sequence of SEQ ID NO: 4. 
     
     
         11 . The gene edited livestock animal of  claim 1 , wherein the genome edited eIF4GI gene has the nucleotide sequence of SEQ ID NO: 5. 
     
     
         12 . The gene edited livestock animal of  claim 1 , wherein the genome edited eIF4GI gene has the nucleotide sequence of SEQ ID NO: 6. 
     
     
         13 . The gene edited livestock animal of  claim 1 , wherein the genome edited eIF4GI gene has the nucleotide sequence of SEQ ID NO: 7. 
     
     
         14 . The gene edited livestock animal of  claim 1 , wherein the genome edited eIF4GI gene has the nucleotide sequence of SEQ ID NO: 8. 
     
     
         15 . The gene edited livestock animal of  claim 1 , wherein the genome edited eIF4GI gene has the nucleotide sequence of SEQ ID NO: 9. 
     
     
         16 . The gene edited livestock animal of  claim 1 , wherein the genome edited eIF4GI gene has the nucleotide sequence of SEQ ID NO: 10. 
     
     
         17 . The gene edited livestock animal of  claim 1 , wherein the genome edited eIF4GI gene has the nucleotide sequence of SEQ ID NO: 11. 
     
     
         18 . The gene edited livestock animal of  claim 1 , wherein the genome edited eIF4GI gene is the only edit to the genome of the livestock animal. 
     
     
         19 . A method of producing a gene edited cow or pig whose genome comprises a genome edited eIF4GI gene, wherein the genome edited eIF4GI gene expresses an altered eIF4GI protein relative to a wild type eIF4GI protein such that the gene edited cow or pig is resistant to cleavage by a proteinase of foot-and-mouth disease virus enzyme , the method comprising the steps of: (a) introducing into a cow or pig fibroblast (i) a TALEN pair that specifically binds to the endogenous eIF4GI gene and causes a double-stranded DNA break to inactivate the eIF4GI gene in the cow or pig fibroblast and (ii) a Homology-dependent repair (HDR) template; (b) permitting to occur homologous recombination events in the cow or pig fibroblast; (c) transferring the transfected fibroblast into an enucleated cow or pig recipient oocyte to generate a transgenic nuclear transfer embryo and activating the nuclear transfer embryo; and (d) transplanting the nuclear transfer embryo into a surrogate mother cow or pig and permitting the implanted embryo to develop such that the transgenic cow or pig whose genome comprises a genome edited eIF4GI gene is produced, wherein the cow or pig expresses the genome edited eIF4GI gene and is resistant to cleavage by the leader proteinase. 
     
     
         20 . The method of  claim 19 , wherein the genome edited eIF4GI gene is the only edit to the genome of the cow or pig fibroblast. 
     
     
         21 . The method of  claim 19 , wherein the genome edited eIF4GI gene has the nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. 
     
     
         22 . The method of  claim 19 , wherein the genome edited eIF4GI gene has the nucleotide sequence of SEQ ID NO: 2 and the livestock animal is cow. 
     
     
         23 . The method of  claim 19 , wherein the altered eIF4GI protein is edited to prevent binding of (i) the leader proteinase (L Pro ), (ii) 3C protease (C Pro ), or (iii) L Pro  and C Pro  of foot-and-mouth disease virus enzyme.