Reconstruction of site specific nuclease binding sites

Abstract

Disclosed herein are methods and compositions for the repair of site specific nuclease binding sites by targeted integration and/or targeted excision of one or more sequences into a cell.

What is claimed is: 
     
         1 .- 20 . (canceled) 
     
     
         21 . A method for targeted excision of a donor polynucleotide from a plant genome, the method comprising the following steps:
 a. Stably inserting the donor polynucleotide sequence within the plant genome;   b. Introducing a site specific nuclease to bind a site specific nuclease binding site, wherein the site specific nuclease binding site flanks the donor polynucleotide;   c. Cleaving at least one site specific nuclease binding site;   d. Removing the donor polynucleotide sequence from the plant genome;   e. Recombining the site specific nuclease binding sites that flank the donor polynucleotide, wherein the recombined site specific nuclease binding site is identical to the site specific nuclease binding site.   
     
     
         22 . The method of  claim 21 , wherein the site specific nuclease is selected from the group consisting of a Zinc Finger nuclease, a CRISPR nuclease, a TALEN nuclease, or any combination thereof. 
     
     
         23 . The method of  claim 21 , wherein the donor polynucleotide sequence is completely removed from the genome. 
     
     
         24 . The method of  claim 23 , wherein the donor polynucleotide sequence comprises a transgene. 
     
     
         25 . The method of  claim 24 , wherein the transgene encodes a selectable marker. 
     
     
         26 . The method of  claim 21 , wherein the repaired site specific nuclease binding site is repaired via a NHEJ-mediated cellular process. 
     
     
         27 . The method of  claim 21 , wherein the repaired site specific nuclease binding site is greater than 6 bp in length. 
     
     
         28 . The method of  claim 21 , wherein the site specific nuclease binding sites are within 3,000 bp to 4,000 bp of one another. 
     
     
         29 . The method of  claim 21 , wherein the recombined site specific nuclease does not comprise an INDEL. 
     
     
         30 . The method of  claim 21 , wherein the donor polynucleotide that is flanked by the site specific nuclease binding sites comprise either a native genomic sequence or a transgenic sequence within a genome of a eukaryotic organism. 
     
     
         31 . The method of  claim 21 , the method further comprising the steps of:
 a. Targeting the recombined site specific nuclease binding site with the site specific nuclease;   b. Cleaving the recombined site specific nuclease binding site with the site specific nuclease;   c. Introducing a second donor polynucleotide sequence;   d. Integrating the second donor polynucleotide sequence within the cleaved site specific nuclease binding site; and   e. Producing a genome comprising the second donor polynucleotide sequence stably integrated within the plant genome.   
     
     
         32 . The method of  claim 21 , wherein the donor polynucleotide sequence comprises a polynucleotide encoding a transgene or a gene expression cassette. 
     
     
         33 . The method of  claim 21 , wherein the site specific nuclease binding site is palindromic. 
     
     
         34 . The method of  claim 21 , wherein the site specific nuclease binding site is non-palindromic. 
     
     
         35 . The method of  claim 21 , wherein the recombined site specific nuclease binding site is inherited in a progeny.