Zinc finger nuclease-mediated homologous recombination
First Claim
Patent Images
1. A plant cell comprising a targeting vector, the targeting vector comprising in a 5′
- to 3′
orientation;
(a) a first region of homology comprising sequences having homology to a region of interest in the plant cell genome;
(b) a first target site for a first zinc finger nuclease, wherein the first target site is not present in the plant cell genome;
(c) a first coding sequence encoding a 5′
piece of a first selectable marker gene, wherein the selectable marker gene is divided into two pieces, a 5′
piece and a 3′
piece, wherein the 3′
end of the 5′
piece and the 5′
end of the 3′
piece are partially duplicative of each other;
(d) a promoter operably linked to a second marker gene, wherein in between the promoter and the second marker gene is a second target site for a second zinc finger nuclease as described in part (e) that follows;
(e) a second target site for a second zinc finger nuclease, wherein the second target site for the second zinc finger nuclease is not present in the plant cell genome;
(f) the second selectable marker gene;
(g) a second coding sequence encoding the 3′
piece of the first selectable marker gene;
(h) the first target site; and
(i) a second region of homology having homology to a region of interest in the plant cell genome,wherein the first marker gene is reconstituted upon cleavage of the second target site by the second zinc finger nuclease.
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Accused Products
Abstract
Disclosed herein are methods and compositions for targeted integration of an exogenous sequence into a predetermined target site in a plant genome.
12 Citations
5 Claims
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1. A plant cell comprising a targeting vector, the targeting vector comprising in a 5′
- to 3′
orientation;(a) a first region of homology comprising sequences having homology to a region of interest in the plant cell genome; (b) a first target site for a first zinc finger nuclease, wherein the first target site is not present in the plant cell genome; (c) a first coding sequence encoding a 5′
piece of a first selectable marker gene, wherein the selectable marker gene is divided into two pieces, a 5′
piece and a 3′
piece, wherein the 3′
end of the 5′
piece and the 5′
end of the 3′
piece are partially duplicative of each other;(d) a promoter operably linked to a second marker gene, wherein in between the promoter and the second marker gene is a second target site for a second zinc finger nuclease as described in part (e) that follows; (e) a second target site for a second zinc finger nuclease, wherein the second target site for the second zinc finger nuclease is not present in the plant cell genome; (f) the second selectable marker gene; (g) a second coding sequence encoding the 3′
piece of the first selectable marker gene;(h) the first target site; and (i) a second region of homology having homology to a region of interest in the plant cell genome, wherein the first marker gene is reconstituted upon cleavage of the second target site by the second zinc finger nuclease. - View Dependent Claims (2, 3, 4, 5)
- to 3′
Specification