Altered Recombinases for Genome Modification

Abstract

The present invention describes methods of identifying altered recombinases and compositions thereof, wherein at least one amino acid is different from a parent, wild-type recombinase and the altered recombinase has improved recombination efficiency towards wild-type and/or pseudo att site sequences relative to the parent, wild-type recombinase. The present invention also includes methods of modifying the genomes of cells using the altered recombinases, including methods of site-specifically integrating a polynucleotide sequence of interest in a genome of a eucaryotic cell.

1 - 22 . (canceled) 
     
     
         23 . An altered, unidirectional site-specific bacteriophage integrase comprising an amino acid sequence having at least 80% sequence identity to one or more of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or the consensus sequence thereof, with integrase activity. 
     
     
         24 . The integrase of  claim 23  having an amino acid sequence selected from the group of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or the consensus sequence thereof. 
     
     
         25 . A nucleic acid sequence encoding the altered, unidirectional site-specific bacteriophage integrase of  claim 23 . 
     
     
         26 . A method of site-specifically integrating a nucleic acid into a genome of a cell, said method comprising:
 a. introducing a targeting vector comprising said nucleic acid and a vector attachment site into a cell;   b. introducing an altered, unidirectional site-specific bacteriophage integrase into said cell; and   c. maintaining said cell under conditions sufficient for said vector to integrate into said genome of said cell by a recombination event mediated by said altered unidirectional site-specific bacteriophage integrase, wherein said unidirectional site-specific bacteriophage integrase comprises an amino acid sequence having at least 80% sequence identity to one or more of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or the consensus sequence thereof, and wherein said integrase has integrase activity.   
     
     
         27 . The method of integration of  claim 26  wherein the cell forms part of a multicellular organism. 
     
     
         28 . The method of integration of  claim 27  wherein the cell forms part of a mammalian multicellular organism. 
     
     
         29 . The method of integration of  claim 26  wherein the integration occurs in vivo. 
     
     
         30 . The method of integration of  claim 26  wherein the targeting vector further comprises a nucleic acid encoding the altered, unidirectional site-specific bacteriophage integrase. 
     
     
         31 . The method of integration of  claim 26  wherein the altered, unidirectional site-specific bacteriophage integrase is introduced into said cell as a nucleic acid encoding said integrase. 
     
     
         32 . The method of integration of  claim 26  wherein the altered, unidirectional site-specific bacteriophage integrase is introduced into said cell in protein form. 
     
     
         33 . A method of site-specifically integrating a nucleic acid into a genome of a cell, said method comprising:
 a. introducing a targeting vector comprising said nucleic acid and a vector attachment site into a cell;   b. introducing an altered, unidirectional site-specific bacteriophage integrase into said cell; and   c. maintaining said cell under conditions sufficient for said vector to integrate into said genome of said cell by a recombination event mediated by said altered unidirectional site-specific bacteriophage integrase, wherein the nucleic acid is integrated into the genome at a site comprising a sequence having at least 40% sequence identity to the approximately 40 base pair nucleic acid sequence surrounding the core TTG nucleic acid sequence of SEQ ID NO:33 or SEQ ID NO:34.   
     
     
         34 . The method of integration of  claim 33  wherein the nucleic acid is integrated into the genome at a site comprising SEQ ID NO:33 or SEQ ID NO:34. 
     
     
         35 . An altered, unidirectional site-specific bacteriophage integrase that is capable of integrating a nucleic acid into a genome of a eukaryotic cell at a site comprising a sequence having at least 40% sequence identity to SEQ ID NO:33 or SEQ ID NO:34. 
     
     
         36 . The altered, unidirectional site-specific bacteriophage integrase of  claim 35  that is capable of integrating a nucleic acid into a genome of a eukaryotic cell at a site comprising SEQ ID NO:33 or SEQ ID NO:34. 
     
     
         37 . A nucleic acid sequence encoding the altered, unidirectional site-specific bacteriophage integrase of  claim 33 . 
     
     
         38 . A kit for use in integrating a nucleic acid into a genome of a cell of a multicellular organism, said kit comprising: (a) a targeting vector comprising said nucleic acid and a vector attachment site; (b) an altered, unidirectional site-specific bacteriophage integrase or a nucleic acid encoding the same, wherein said unidirectional site-specific bacteriophage integrase has at least 80% sequence identity to SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or the consensus sequence thereof, wherein said integrase has integrase activity.