Engineered zinc finger proteins targeting 5-enolpyruvyl shikimate-3-phosphate synthase genes
The present disclosure relates to engineered zinc finger proteins that target 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) genes in plants and methods of using such zinc finger proteins in modulating gene expression, gene inactivation, and targeted gene modification. In particular, the disclosure pertains to zinc finger nucleases for targeted cleavage and alteration of EPSPS genes.
1. A method for stimulating homologous recombination in the genome of a plant cell, the method comprising the step of:
introducing a zinc finger nuclease (ZFN) into the plant cell in the presence of a DNA segment in the plant cell, wherein the DNA segment comprises at least one target site in an 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene, wherein said target site comprises any one of SEQ ID NO: 141-153, wherein the ZFN cleaves the DNA segment and stimulates homologous recombination, wherein the ZFN comprises a non-naturally occurring zinc finger protein (ZFP) that binds to said target site in the EPSPS gene, wherein the ZFP comprises the recognition helix regions designated and ordered as set forth in any single row of Table A.
2. The method of claim 1 , wherein the homologous recombination results in a deletion within the DNA segment.
3. The method of claim 2 , wherein the deleted DNA sequence is replaced by an exogenous sequence, the method further comprising:
introducing a polynucleotide comprising the exogenous sequence into the cell, wherein the polynucleotide comprises at least a first and a second region of homology to the EPSPS gene, said regions of homology flanking the exogenous sequence.
4. The method of claim 3 , wherein the exogenous sequence encodes a selectable marker.
5. The method of claim 4 , wherein the selectable marker is selected from the group consisting of green fluorescent protein, β-glucuronidase, phosphinothricin N-acetyl transferase, neomycin phosphotransferase, hygromycin phosphotransferase, β-lactamase, catechol dioxygenase, α-amylase, tyrosinase, β-galactosidase, luciferase, aequorin, nitrilase, acetolactate synthase, dihydrofolate reductase, dalapon dehalogenase, and anthranilate synthase.
6. The method of claim 3 , wherein the exogenous sequence is an EPSPS gene sequence.
7. The method of claim 6 wherein the EPSPS gene sequence comprises a mutation relative to the wild-type EPSPS sequence.
8. The method of claim 7 , wherein the mutation increases tolerance of a plant to herbicide glyphosate relative to a control plant.