Methods and compositions for targeted single-stranded cleavage and targeted integration

Abstract

Disclosed herein are methods and compositions for generating a single-stranded break in a target sequence, which facilitates targeted integration of one or more exogenous sequences.

1 .- 11 . (canceled) 
     
     
         12 . A method of inserting an exogenous sequence into a region of interest in a cell, the method comprising:
 generating a single-stranded break in the region of interest by providing a protein complex including a fusion protein comprising a DNA-binding domain and at least one catalytically inactive cleavage domain or cleavage half-domain to the cell; and   inserting the exogenous sequence into the single stranded break in the region of interest, thereby inserting the exogenous sequence into the region of interest in the cell.   
     
     
         13 . The method of  claim 12 , wherein the exogenous sequence replaces a wild-type genomic sequence. 
     
     
         14 . The method of  claim 12 , wherein the insertion of the exogenous sequence partially or fully inactivates a target sequence within the region of interest. 
     
     
         15 . The method of  claim 12 , wherein the protein complex further comprises a second fusion protein comprising a zinc finger domain and a catalytically active cleavage half domain and the catalytically inactive cleavage half domain forms a heterodimer with the catalytically active cleavage half-domain of the second fusion protein. 
     
     
         16 . The method of  claim 15 , wherein the heterodimer generates the single-stranded break in the region of interest. 
     
     
         17 . The method of  claim 12 , wherein the region of interest is a double-stranded sequence. 
     
     
         18 . The method of  claim 17 , wherein the double-stranded sequence is cellular chromatin. 
     
     
         19 . A method for replacement of a region of interest in a cell with a first nucleotide sequence comprising: (a) engineering a first zinc finger binding domain to bind to a second sequence in the region of interest; (b) providing a second zinc finger binding domain to bind to a third sequence in the region of interest; (c) expressing a first fusion protein in a cell, the first fusion protein comprising the first zinc finger binding domain and a first catalytically active cleavage half-domain; (d) expressing a second fusion protein in the cell, the second fusion protein comprising the second zinc finger binding domain and a second catalytically inactive cleavage half-domain; and (e) contacting the cell with a polynucleotide comprising the first nucleotide sequence. 
     
     
         20 . The method of  claim 19 , wherein the region of interest comprises cellular chromatin. 
     
     
         21 . The method of  claim 19 , wherein the first fusion protein binds to the second sequence and the second fusion protein binds to the third sequence, thereby positioning the cleavage half-domains such that a single-stranded break is made in cellular chromatin in the region of interest and a nucleotide sequence in the region of interest is replaced with the first nucleotide sequence. 
     
     
         22 . The method of  claim 21 , wherein the single-stranded break in cellular chromatin is made in the region of interest at a site between the second and third sequences. 
     
     
         23 . The method of  claim 19 , wherein the zinc finger nucleases are provided to the cells as proteins and/or as one or more polynucleotides encoding said zinc finger nucleases. 
     
     
         24 . The method of  claim 23 , wherein a single polynucleotide comprises sequences encoding both the first and the second fusion protein.