Targeted chromosomal mutagenesis using zinc finger nucleases

Abstract

The present invention provides for a method or methods of targeted genetic recombination or mutagenesis in a host cell or organism, and compositions useful for carrying out the method. The targeting method of the present invention exploits endogenous cellular mechanisms for homologous recombination and repair of double stranded breaks in genetic material. The present invention provides numerous improvements over previous mutagenesis methods, such advantages include that the method is generally applicable to a wide variety of organisms, the method is targeted so that the disadvantages associated with random insertion of DNA into host genetic material are eliminated, and certain embodiments require relatively little manipulation of the host genetic material for success. Additionally, it provides a method that produces organisms with specific gene modifications in a short period of time.

1 .- 98 . (canceled) 
     
     
         99 . A plant cell comprising a mutation introduced by a Zinc Finger Nuclease (ZFN) that binds to a target site in a chosen host chromosomal target locus; wherein the mutation comprises an insertion and/or deletion of genetic material surrounding the target site. 
     
     
         100 . The plant cell of  claim 99 , wherein the mutation is an insertion of genetic material. 
     
     
         101 . The plant cell of  claim 99 , wherein the mutation is a deletion of genetic material. 
     
     
         102 . The plant cell of  claim 99 , wherein the mutation is both a deletion and an insertion of genetic material. 
     
     
         103 . The plant cell of  claim 99 , wherein a donor DNA is inserted into the chromosomal target locus. 
     
     
         104 . The plant cell of  claim 103 , wherein the donor DNA provides a gene sequence that encodes a product to be produced in the plant cell. 
     
     
         105 . The plant cell of  claim 103 , wherein the donor DNA provides a gene sequence that encodes a pharmaceutical, hormone, protein, nutriceutical or chemical. 
     
     
         106 . The plant cell of  claim 99 , wherein the method further comprises: selecting a zinc finger nuclease comprises a zinc finger protein DNA binding domain capable and a non-specific DNA cleavage domain capable of cleaving double-stranded DNA when operatively linked to said DNA-binding domain. 
     
     
         107 . The plant cell of  claim 106 , wherein the DNA binding domain is comprised of three zinc fingers. 
     
     
         108 . The plant cell of  claim 106 , wherein the zinc finger DNA binding domain is a Cis2His2 zinc finger. 
     
     
         109 . The plant cell of  claim 106 , wherein the cleavage domain is from a Type II restriction endonuclease. 
     
     
         110 . The plant cell of  claim 109 , wherein the Type II restriction endonuclease is Fold. 
     
     
         111 . The plant cell of  claim 104 , wherein the gene sequences encodes one or more selectable markers. 
     
     
         112 . The plant cell of  claim 111 , wherein the one or more selectable markers provides positive selection for cells expressing the marker. 
     
     
         113 . The plant cell of  claim 111 , wherein the one or more selectable markers provides negative selection for cells expressing the marker. 
     
     
         114 . The plant cell of  claim 111 , wherein the selectable marker provides positive and negative selection for cells expressing the marker. 
     
     
         115 . The plant cell of  claim 104 , wherein the donor DNA comprises a constitutively active or inducible promoter upstream of the gene sequence that encodes a product to be produced in the plant cell.