Genome editing in rats using zinc-finger nucleases

Abstract

Disclosed herein are methods and compositions for genome editing of one or more loci in a rat, using fusion proteins comprising a zinc-finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding said fusion proteins are also provided, as are cells comprising said polynucleotides and fusion proteins.

What is claimed is: 
     
         1 . A method for modifying one or more endogenous cellular genes in a rat cell, the method comprising: introducing, into the rat cell, one or more polynucleotides encoding one or more zinc finger nucleases (ZFNs) that bind to a target site in the one or more genes under conditions such that the ZFN(s) is (are) expressed and the one or more endogenous cellular genes are cleaved and modified. 
     
     
         2 . The method of  claim 1 , wherein the modification comprises introducing an exogenous sequence into the genome of a rat cell by homologous recombination stimulated by cleavage of the one or more endogenous cellular genes. 
     
     
         3 . The method of  claim 2 , wherein the exogenous sequence is integrated physically into the genome. 
     
     
         4 . The method of  claim 2 , wherein the exogenous sequence is integrated into the genome by copying of the exogenous sequence into the host cell genome via nucleic acid replication processes. 
     
     
         5 . The method of  claim 4 , wherein the nucleic acid replication process comprises homology-directed repair of the double strand break. 
     
     
         6 . The method of  claim 4 , wherein the nucleic acid replication process comprises non-homology dependent targeted integration. 
     
     
         7 . The method of  claim 1 , wherein the modification results from non-homologous end joining following cleavage. 
     
     
         8 . The method of  claim 7 , wherein first and second zinc finger nucleases cleave the genome at two sites and further wherein the non-homologous end joining results in a deletion between the first and second cleavage sites. 
     
     
         9 . The method of  claim 1 , wherein the zinc finger nucleases comprise a cleavage domain or cleavage half-domain of a Type IIS restriction endonuclease. 
     
     
         10 . A method for germline disruption of one or more target genes in rat, the method comprising modifying one or more gene sequences in the genome of one or more cells of a rat embryo the method comprising: modifying one or more of the target genes in one or more cells of a rat embryo according to the method of  claim 1 ; and allowing the rat embryo to develop, wherein that the modified gene sequences are present in at least a portion of gametes of the sexually mature rat. 
     
     
         11 . The method of  claim 10 , wherein the modification comprises integration of an exogenous sequence into the genome of a rat cell by homologous recombination stimulated by cleavage of the one or more endogenous cellular genes. 
     
     
         12 . The method of  claim 10 , wherein the modification results from non-homologous end joining following cleavage. 
     
     
         13 . A method of creating one or more heritable mutant alleles in rat loci of interest, the method comprising modifying one or more loci in the genome of one or more cells of a rat embryo by the method of  claim 1 ; raising the rat embryo to sexual maturity; and allowing the sexually mature rat to produce offspring; wherein at least some of the offspring comprise the mutant alleles. 
     
     
         14 . The method of  claim 13 , wherein the modification comprises integration of an exogenous sequence into the genome of a rat cell by homologous recombination stimulated by cleavage of the one or more endogenous cellular genes. 
     
     
         15 . The method of  claim 13 , wherein the modification results from non-homologous end joining following cleavage. 
     
     
         16 . A method for making a deletion in an endogenous IgM gene, the method comprising:
 (i) introducing mRNA into a rat embryo, wherein the mRNA comprises (i) mRNA encoding a first zinc finger nuclease (ZFN) that binds to a first target site, wherein the first ZFN comprises a cleavage domain and a zinc finger protein, wherein the first zinc finger protein-comprises the following recognition helices in the following order:   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 41) 
                 
                     
                   DRSHLTR; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 40) 
                 
                     
                   RSDALTQ; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 28) 
                 
                     
                   DRSDLSR; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 39) 
                 
                     
                   RSDALAR; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 38) 
                 
                     
                   RSDSLSA;  
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 37) 
                 
                     
                   TSSNRKT; 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       and
 (ii) mRNA encoding a second ZFN that binds to a second target site in the endogenous rat IgM gene, wherein the second ZFN comprises a cleavage domain and a zinc finger protein comprising the following recognition helices in the following order: 
 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 46) 
                 
                     
                   NKVGLIE; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 45) 
                 
                     
                   TSSDLSR; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 44) 
                 
                     
                   RSDHLSR; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 43) 
                 
                     
                   RSDNLSE;  
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 42) 
                 
                     
                   QNAHRKT; 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       such that the first and second ZFNs are expressed, dimerize and cleave the endogenous rat IgM gene and non-homologous end joining occurs causing the cell to have a deletion in the endogenous rat IgM gene. 
     
     
         17 . The method of  claim 16 , wherein the cleavage domain is a Type IIS restriction endonuclease cleavage domain.