Compositions and methods for enhancing oligonucleotide-directed nucleic acid sequence alteration

Abstract

Composition and methods for enhancing oligonucleotide-directed nucleic acid sequence alteration in vivo, ex vivo and in vitro are presented. These methods and compositions involve cells and cell-free extracts with altered levels or activities of a protein from the RAD52 epistasis group, the mismatch repair group and/or the excision repair group.

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         3 . The method of  claim 19  in which the nucleic acid sequence alteration is a deletion, insertion or replacement alteration.  
     
     
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         7 . The method of  claim 19 , wherein the nucleic acid sequence alteration affects 1, 2 or 3 consecutive nucleotides in a target nucleic acid.  
     
     
         8 . The method of  claim 19 , wherein the oligonucleotide binds to the non-transcribed strand of a gene at a target site.  
     
     
         9 . The method of  claim 19 , wherein the oligonucleotide is a chimeric, RNA-DNA, double-hairpin oligonucleotide or a modified single-stranded oligonucleotide.  
     
     
         10 . The method of  claim 19 , wherein the nucleic acid sequence alteration is in a target nucleic acid selected from the group consisting of: plasmids, cosmids, artificial chromosomes, YACs, BACs, PLACs and BiBACs.  
     
     
         11 . The method of  claim 19 , wherein the cell is selected from a fungal, plant, animal, mammal or human cell.  
     
     
         12 . The method of  claim 19 , wherein the cell-free extract is from a fungal, plant, animal, mammal or human cell.  
     
     
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         19 . A method of oligonucleotide-mediated nucleic acid sequence alteration comprising: 
 providing an altered cell or cell-free extract comprising a target nucleic acid sequence wherein the altered cell or cell-free extract has been altered such that it exhibits increased expression or protein activity relative to unaltered cell or cell-free extract of at least one protein selected from the group consisting of RAD10, RAD51, RAD52, RAD54, RAD55, MRE11, PMS1, XRS2, and homologs and combinations thereof;    providing an oligonucleotide adapted for altering at least one nucleotide in said target nucleic acid sequence; and    introducing said oligonucleotide to the cell or cell-free extract under suitable conditions to allow recombination with the target nucleic acid sequence.