Compositions and methods for enhancing homologous recombination

Abstract

The present disclosure generally relates to compositions and methods for improving the efficiency of homologous recombination. In particular, the disclosure relates to reagents and the use of such reagents.

1 . A method for performing homologous recombination, the method comprising:
 (a) generating a double-stranded break in a nucleic acid molecule present inside a cell to produce a cleaved nucleic acid molecule, and   (b) contacting the cleaved nucleic acid molecule generated in (a) with a donor nucleic acid molecule,   wherein the cleaved nucleic acid molecule and the donor nucleic acid molecule each contain matched termini on at least one end,   wherein the matched termini on at least one end of the cleaved nucleic acid molecule and the donor nucleic acid molecule is at least ten nucleotides in length, and   wherein the matched region of the cleaved nucleic acid molecule is single-stranded or double-stranded and the matched region of the donor nucleic acid molecule is single-stranded.   
     
     
         2 . The method of  claim 1 , wherein the matched termini on at least one end of the cleaved nucleic acid molecule and the donor nucleic acid molecule have 5′ overhangs or 3′ overhangs. 
     
     
         3 . The method of  claim 1 , wherein the matched termini on at least one end of the cleaved nucleic acid molecule and the donor nucleic acid molecule have one 5′ overhang and one 3′ overhang. 
     
     
         4 .- 7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the cleaved nucleic acid molecule has at least one terminus with a single-stranded region. 
     
     
         9 . The method of  claim 1 , wherein the double-stranded break in the nucleic acid molecule present inside the cell is generated by the formation of two nicks, one in each strand of the nucleic acid molecule. 
     
     
         10 . The method of  claim 9 , wherein the cleaved nucleic acid molecule has at least one blunt terminus. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein donor nucleic acid molecule contains one or more nuclease resistant groups in at least one strand of at least one terminus. 
     
     
         13 . The method of  claim 12 , wherein donor nucleic acid molecule contains one or more nuclease resistant groups in both strands of both termini 
     
     
         14 .- 15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein the donor nucleic acid molecule has asymmetric termini. 
     
     
         17 .- 30 . (canceled) 
     
     
         31 . A method for performing homologous recombination in a population of cells, the method comprising,
 (a) contacting the population of cells with a nucleic acid cutting entity under conditions that allow for the generation of double-stranded break at a target locus in nucleic acid present inside cells of the population, to produce cells containing an intracellular cleaved nucleic acid molecule, and   (b) introducing a donor nucleic acid molecule into cells generated in step (a) with under conditions that allow for homologous recombination to occur, wherein homologous recombination occurs at the target locus in at least 20% of the cells of the population, and   wherein the target locus and/or the donor nucleic acid molecule have one or more of the following characteristics:   (a) the target locus and the donor nucleic acid molecule share at least one matched terminus,   (b) the donor nucleic acid molecule contains one or more nuclease resistant group,   (c) donor nucleic acid molecule has asymmetric termini,   (d) the target locus cut site is within 15 nucleotides of the location where alteration is desired,   (e) the nucleic acid cutting entity, or components thereof, and the donor nucleic acid molecule are contacted with the cells of the population at different times, and/or   (f) the amount of the donor nucleic acid molecule contacted with cells of the population is in a range that allows for efficient uptake and homologous recombination.   
     
     
         32 .- 39 . (canceled) 
     
     
         40 . The method of  claim 31 , wherein the cells of the population are contacted with the nucleic acid cutting entity, or components thereof, before the cells of the population are contacted with the donor nucleic acid molecule. 
     
     
         41 . The method of  claim 31 , wherein the cells of the population are contacted with the nucleic acid cutting entity, or components thereof, for between 5 and 60 minutes before the cells of the population are contacted with the donor nucleic acid molecule. 
     
     
         42 . The method of  claim 31 , wherein the donor nucleic acid molecule contains one or more nuclease resistant group at one or more terminus. 
     
     
         43 .- 46 . (canceled) 
     
     
         47 . The method of  claim 31 , wherein the target locus cut site is within 10 nucleotides of the location where alteration is desired. 
     
     
         48 . The method of  claim 31 , wherein the target locus cut site comprises a single stranded region that includes all or part of the location where alteration is desired. 
     
     
         49 . The method of  claim 31 , wherein the single-stranded region contains a single mismatched nucleotide between the target locus and the donor nucleic acid molecule. 
     
     
         50 . The method of  claim 31 , wherein the amount of donor nucleic acid is between 50 and 900 ng per 1×10 5  cells. 
     
     
         51 . The method of  claim 31 , wherein donor nucleic acid molecules are introduced into cells of the population by electroporation or transfection. 
     
     
         52 . A method for performing homologous recombination in a cell, the method comprising:
 (a) introducing into the cell a nucleic acid cutting entity capable of generating a double-stranded break at a specified location in a nucleic acid molecule present inside a cell to produce a cleaved nucleic acid molecule, and   (b) introducing a donor nucleic acid molecule into the cell,   wherein step (a) is performed before step (b) or wherein step (b) is performed before step (a).   
     
     
         53 . The method of  claim 52 , wherein the introduction of the nucleic acid cutting entity or the donor nucleic acid molecule into the cell is mediated by electroporation.