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 . A method of identifying an altered recombinase, said method comprising
 providing a population of cells, each cell of said population comprising a first plasmid, said first plasmid comprising a promoter adjacent a first recombination site adjacent a transcription terminator adjacent a second recombination site adjacent a coding sequence of interest, such that the order of these components is promoter-first recombination site-transcription terminator-second recombination site-coding sequence of interest, wherein said first and second recombination sites act as substrates for a first recombinase;   transforming said population of cells with a group of second plasmids, said group comprising at least one second plasmid comprising a coding sequence for an altered recombinase operably linked to a promoter functional in said cell, said coding sequence for the altered recombinase differing from the coding sequence of said first recombinase by at least one base pair;   maintaining said cells under conditions that allow recombination to occur between said first and second recombination sites, wherein said recombination is mediated by said altered recombinase;   screening said population of transformed cells for production of a product encoded by said coding sequence of interest;   isolating said transformed cells that produce said product; and   identifying a coding sequence of an altered recombinase encoded within said transformed cells.   
     
     
         2 . The method of  claim 1 , wherein said altered recombinase provides an improved recombination frequency between said first and second recombination sites relative to the recombination frequency between said first and second recombination sites mediated by said first recombinase. 
     
     
         3 . The method of  claim 1 , wherein said first recombinase is a wild-type phage recombinase selected from the group consisting of NC31, TP901-1, and R4. 
     
     
         4 . The method of  claim 3 , wherein said first recombinase is NC31. 
     
     
         5 . The method of  claim 4 , wherein said first or second recombination site is a wild-type attB or an attP substrate. 
     
     
         6 . The method of  claim 4 , wherein said first or second recombination site is a pseudo-attB or a pseudo-attP. 
     
     
         7 . The method of  claim 6 , wherein said pseudo-attB or pseudo-attP is derived from a sequence native to a human genome. 
     
     
         8 . The method of  claim 6 , wherein said pseudo-attB or pseudo-attP is derived from a sequence native to a rodent genome. 
     
     
         9 . The method of  claim 3 , wherein said first recombinase is R4. 
     
     
         10 . The method of  claim 9 , wherein said first or second recombination site is a wild-type attB or an attP substrate. 
     
     
         11 . The method of  claim 4 , wherein said first or second recombination site is a pseudo-attB or a pseudo-attP. 
     
     
         12 . The method of  claim 1 , wherein said cells are bacterial cells. 
     
     
         13 . The method of  claim 1 , wherein said coding sequence of interest encodes a polypeptide product. 
     
     
         14 . The method of  claim 13 , wherein said coding sequence of interest is a beta-galactosidase coding sequence. 
     
     
         15 . The method of  claim 1 , wherein said coding sequence of interest encodes a selectable marker and said screening includes a selection based on expression of the selectable marker. 
     
     
         16 . An altered recombinase produced by the method of  claim 1 , wherein said altered recombinase comprises a polypeptide wherein at least one amino acid is different from a wild-type sequence of the first recombinase, wherein the altered recombinase has improved recombination efficiency towards wild-type or pseudo att site sequences relative to the first recombinase. 
     
     
         17 . An altered recombinase consisting of a polypeptide sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. 
     
     
         18 . A nucleic acid sequence encoding the altered recombinase of  claim 17 . 
     
     
         19 . A method of site-specifically integrating a polynucleotide sequence of interest in a genome of a cell, said method comprising:
 introducing (i) a circular targeting construct, comprising a first recombination site and the polynucleotide sequence of interest, and (ii) an altered, site-specific recombinase of  claim 16  into the cell, wherein the genome of said cell comprises a second recombination site native to the genome and recombination between the first and second recombination sites is facilitated by the altered recombinase,   maintaining the cell under conditions that allow recombination between said first and second recombination sites, wherein the recombination is mediated by the altered recombinase and the result of the recombination is site-specific integration of the polynucleotide sequence of interest in the genome of the cell.   
     
     
         20 . The method of  claim 19 , wherein said altered recombinase consists of a polypeptide sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. 
     
     
         21 . The method of  claim 19 , wherein the altered recombinase is introduced into the cell as a polypeptide. 
     
     
         22 . The method of  claim 19 , wherein the altered recombinase is introduced into the cell as a polynucleotide encoding the altered recombinase.