Targeted deletion of cellular dna sequences

Abstract

Disclosed herein are methods and compositions for targeted deletion of double-stranded DNA. The compositions include fusion proteins comprising a cleavage domain (or cleavage half-domain) and an engineered zinc finger domain, and polynucleotides encoding same. Methods for targeted deletion include introduction of such fusion proteins, or polynucleotides encoding same, into a cell such that two targeted cleavage events occur. Subsequent cellular repair mechanisms result in deletion of sequences between the two cleavage sites.

What is claimed is: 
     
         1 . A method for deleting a region of interest in an endogenous gene in a plant cell, the method comprising:
 cleaving the genome at first and second cleavage sites surrounding the region of interest using first and second pairs of artificial zinc finger nucleases that bind to first, second, third and fourth target sites in the endogenous genome, each zinc finger nuclease comprising:   a fusion protein comprising an engineered zinc finger protein DNA-binding domain that binds to the target site in the endogenous genome;   and a FokI nuclease domain,   wherein each target site comprises at least 3 target subsites and the first cleavage site lies between the first and second target sites and the second cleavage site lies between the third and fourth target sites;   such that region of interest is deleted from the genome and further wherein at least two of the target sites are maintained in the endogenous genome following deletion of the region of interest.   
     
     
         2 . The method of  claim 1 , wherein the first and second target sites are separated by 2 to 50 nucleotides. 
     
     
         3 . The method of  claim 1 , wherein the region of interest comprises at least 100 base pairs. 
     
     
         4 . The method of  claim 1 , wherein the region of interest comprises at least 400 base pairs. 
     
     
         5 . The method of  claim 1 , wherein the region of interest comprises at least 1000 base pairs. 
     
     
         6 . The method of  claim 1 , wherein the plant cell is from a monocotyledonous and dicotyledonous plant. 
     
     
         7 . The method of  claim 1 , wherein the plant cell is a crop plant cell selected from the group consisting of a grain crop, a fruit crop, a forage crop, a vegetable crop, an oil crop, a flowering plant crop and an experimental crop. 
     
     
         8 . The method of  claim 1 , wherein the plant cell is a seed cell. 
     
     
         9 . A plant generated from the plant cell produced by the method of  claim 1 .