TARGETED CHROMOSOMAL MUTAGENESIS USING ZINC FINGER NUCLEASES
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 . A plant cell comprising a mutation introduced by a Zinc Finger Nuclease (ZFN) that binds to an endogenous target site in a chosen host chromosomal target locus of the plant cell; wherein the mutation comprises exogenous donor DNA inserted into the host chromosomal target locus of the plant cell.
2 . The plant cell of claim 1 , wherein ZFN cleaves an endogenous target site in the chosen host chromosomal target locus of the plant cell.
3 . The plant cell of claim 1 , wherein the exogenous donor DNA provides a gene sequence that encodes a product to be produced in the plant cell.
4 . The plant cell of claim 1 , wherein the exogenous donor DNA provides a gene sequence that encodes a pharmaceutical, hormone, protein, nutraceutical or chemical.
5 . The plant cell of claim 1 , wherein the exogenous donor DNA encodes one or more selectable markers.
6 . The plant cell of claim 5 , wherein the one or more selectable markers provides positive selection for plant cells expressing the marker.
7 . The plant cell of claim 5 , wherein the one or more selectable markers provides negative selection for plant cells expressing the marker.
8 . The plant cell of claim 5 , wherein the one or more selectable marker provides positive and negative selection for plant cells expressing the marker.
9 . The plant cell of claim 3 , wherein the exogenous donor DNA comprises a constitutively active or inducible promoter upstream of the gene sequence that encodes the product to be produced in the plant cell.
10 . The plant cell of claim 1 , further comprising a nucleic acid molecule encoding a chimeric zinc finger nuclease.
11 . The plant cell of claim 10 , wherein the chimeric zinc finger nuclease comprises a zinc finger protein DNA binding domain capable of cleaving double-stranded DNA.
12 . A composition comprising a plant cell of claim 1 , wherein the composition further comprises a nucleic acid construct comprising a DNA encoding a chimeric zinc finger nuclease.
13 . The composition of claim 12 , wherein the chimeric zinc finger nuclease comprises a zinc finger protein DNA binding domain capable of cleaving double-stranded DNA.
14 . A plant cell or whole plant regenerated from the plant cell of claim 1 .
15 . A plant cell or whole plant regenerated from the plant cell of claim 10 .