Chromosomal Modification Involving the Induction of Double-Stranded DNA Cleavage and Homologous Recombination at the Cleavage Site

Abstract

Methods of modifying, repairing, attenuating and inactivating a gene or other chromosomal DNA in a cell are disclosed. Also disclosed are methods of treating or prophylaxis of a genetic disease in an individual in need thereof. Further disclosed are chimeric restriction endonucleases.

1 . A method for treating or prophylaxis of a genetic disease in an individual in need thereof comprising inducing in cells of the individual double stranded cleavage at a site of interest using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain, under conditions appropriate for chromosomal DNA homologous to the region surrounding the site of interest to be introduced into the site of interest and repair of the site of interest. 
     
     
         2 . The method of  claim 1  wherein the DNA binding sequence is a zinc finger binding domain. 
     
     
         3 . The method of  claim 1  wherein the DNA binding sequence is a meganuclease recognition site. 
     
     
         4 . The method of  claim 1  wherein the DNA cleavage domain is a restriction endonuclease cleavage domain. 
     
     
         5 . The method of  claim 3  wherein the meganuclease recognition site is a I-SceI recognition site and the DNA cleavage domain is the FokI cleavage domain. 
     
     
         6 . A method of correcting a genetic lesion in chromosomal DNA of a cell comprising inducing in the cell double stranded cleavage at a site of interest in the genetic lesion using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain, under conditions appropriate for chromosomal DNA homologous to the region surrounding the site of interest to be introduced into the site of interest and correction of the genetic lesion. 
     
     
         7 . The method of  claim 6  wherein the DNA binding sequence is a zinc finger binding domain. 
     
     
         8 . The method of  claim 6  wherein the DNA binding sequence is a meganuclease recognition site. 
     
     
         9 . The method of  claim 6  wherein the DNA cleavage domain is a restriction endonuclease cleavage domain. 
     
     
         10 . The method of  claim 8  wherein the meganuclease recognition site is a I-SceI recognition site and the DNA cleavage domain is the FokI cleavage domain. 
     
     
         11 . A method of modifying a specific sequence in chromosomal DNA of a cell comprising inducing in the cell double stranded cleavage at a site of interest in the specific sequence to be modified using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain, under conditions appropriate for chromosomal DNA homologous to the region surrounding the site of interest to be introduced into the site of interest and modification of the specific sequence. 
     
     
         12 . The method of  claim 11  wherein the DNA binding sequence is a zinc finger binding domain. 
     
     
         13 . The method of  claim 11  wherein the DNA binding sequence is a meganuclease recognition site. 
     
     
         14 . The method of  claim 11  wherein the DNA cleavage domain is a restriction endonuclease cleavage domain. 
     
     
         15 . The method of  claim 13  wherein the meganuclease recognition site is a I-SceI recognition site and the DNA cleavage domain is the FokI cleavage domain. 
     
     
         16 . A method of repairing a specific sequence of interest in chromosomal DNA of a cell comprising inducing in the cell double stranded cleavage at a site of interest using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain, under conditions appropriate for chromosomal DNA homologous to the region surrounding the site of interest to be introduced into the site of interest and repair of the specific sequence of interest. 
     
     
         17 . The method of  claim 16  wherein the specific sequence of interest is a mutation. 
     
     
         18 . The method of  claim 16  wherein the DNA binding sequence is a zinc finger binding domain. 
     
     
         19 . The method of  claim 16  wherein the DNA binding sequence is a meganuclease recognition site. 
     
     
         20 . The method of  claim 16  wherein the DNA cleavage domain is a restriction endonuclease cleavage domain. 
     
     
         21 . The method of  claim 19  wherein the meganuclease recognition site is a I-SceI recognition site and the DNA cleavage domain is the FokI cleavage domain. 
     
     
         22 . A chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain. 
     
     
         23 . The chimeric restriction endonuclease of  claim 22  wherein the DNA binding sequence is a zinc finger binding domain. 
     
     
         24 . The chimeric restriction endonuclease of  claim 22  wherein the DNA binding sequence is a meganuclease recognition site. 
     
     
         25 . The chimeric restriction endonuclease of  claim 22  wherein the DNA cleavage domain is a restriction endonuclease cleavage domain. 
     
     
         26 . The chimeric restriction endonuclease of  claim 24  wherein the meganuclease recognition site is a I-SceI recognition site and the DNA cleavage domain is the FokI cleavage domain.