Engineered zinc finger proteins targeting 5-enolpyruvyl shikimate-3-phosphate synthase genes

• US 8,399,218 B2
• Filed: 09/25/2008
• Issued: 03/19/2013
• Est. Priority Date: 09/27/2007
• Status: Active Grant

• Current Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Therapeutics, Inc.
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Biosciences Incorporated
• Inventors: Gupta, Manju, Palta, Asha M., Novak, Stephen, Urnov, Fyodor, Gopalan, Sunita
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US12/284,888
• Publication Number:
  US 20090205083A1
• Time in Patent Office: 1,636 Days
• Field of Search: 435/69.1, 530/350, 536/23.1
• US Class Current: 435/69.1
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 9/1092 3-Phosphoshikimate 1-carboxyvinyltransferase (2.5.1.19), i.e. 5-enolpyruvylshikimate-3-phosphate synthase

Abstract

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.

Claims

1. A non-naturally occurring zinc finger protein (ZFP) that binds to a target site in an endogenous 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene, the zinc finger protein comprising the zinc finger domains set forth in any single row of Table A.

2. The ZFP of claim 1, wherein the ZFP is a fusion protein comprising one or more functional domains.

3. The ZFP of claim 2, wherein the functional domains selected from the group consisting of a transcriptional repressor, a nuclease cleavage domain, a methyl transferase, a histone deacetylase, a transcriptional activator, and a histone acetyltransferase.

4. The ZFP of claim 3, wherein the functional domain is a nuclease cleavage domain.

5. The ZFP of claim 4, wherein the nuclease cleavage domain cleaves one or more EPSPS genes.

6. A polynucleotide encoding one or more zinc finger proteins (ZFPs) according to claim 1.

7. A plant host cell comprising one or more polynucleotides according to claim 6.

8. The plant host cell of claim 7, wherein the cell is stably transfected with the one or more polynucleotides.

9. The plant host cell of claim 7, wherein the cell is transiently transfected with the one or more polynucleotides.

10. A method for cleaving one or more EPSPS genes in a plant cell, the method comprising:

introducing into said plant cell one or more polynucleotides encoding a non-naturally occurring zinc finger protein (ZFP) that binds to a target site in an endogenous 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene, wherein the zinc finger protein comprises the zinc finger domains set forth in any single row of Table A, said ZFP further comprising a nuclease cleavage domain which cleaves one or more EPSPS genes,

expressing the one or more ZFPs in the cell,

wherein the ZFPs cleave one or more EPSPS genes.

11. The method of claim 10, wherein at least one polynucleotide is stably transfected into the plant cell.

12. The method of claim 10, wherein at least one polynucleotide is transiently transfected into the plant cell.

13. The method of claim 10, wherein all EPSPS paralogous genes in the plant cell are cleaved.

14. The method of claim 10, wherein one or more paralogous genes in the plant cell are cleaved.

15. A method for modulating expression of an EPSPS gene in a cell in a plant, the method comprising:

expressing in said cell one or more polynucleotides encoding a non-naturally occurring zinc finger protein (ZFP) that binds to a target site in an endogenous 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene, wherein the zinc finger protein comprises the zinc finger domains set forth in any single row of Table A, said ZFP further comprising a transcriptional activator domain, wherein the ZFP binds to a target sight in the EPSPS gene, thereby increasing expression of the EPSPS gene.

16. The method of claim 15, wherein the expression of the ZFP increases tolerance of a plant to the herbicide glyphosate.

17. A method for modulating expression of an EPSPS gene in a cell in a plant, the method comprising:

expressing in said cell one or more polynucleotides encoding a non-naturally occurring zinc finger protein (ZFP) that binds to a target site in an endogenous 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene, wherein the zinc finger protein comprises the zinc finger domains set forth in any single row of Table A, said ZFP further comprising a transcriptional activator domain, wherein the ZFP binds to a target sight in the EPSPS gene, thereby decreasing expression of the EPSPS gene.

18. The method of claim 17, wherein the expression of the ZFP decreases tolerance of a plant to the herbicide glyphosate.