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

• US 20090205083A1
• Filed: 09/25/2008
• Published: 08/13/2009
• Est. Priority Date: 09/27/2007
• Status: Active Grant

• Current Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Gopalan, Sunita, Palta, Asha M., Gupta, Manju, Urnov, Fyodor, Novak, Stephen
• Granted Patent: US 8,399,218 B2
• Time in Patent Office: Days
• US Class Current: 800/298
• 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 an EPSPS target genomic region of interest, said ZFP comprising one or more engineered zinc finger binding domains.

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, comprising one or more functional domains selected from the group consisting of a transcriptional repressor, an endonuclease, a methyl transferase, a histone deacetylase, a transcriptional activator, and a histone acetyltransferase.

4. The ZFP of claim 3, wherein the ZFP is a zinc finger nuclease (ZFN) that cleaves the EPSPS target genomic region of interest, said ZFN comprising one or more engineered zinc finger binding domains and a nuclease cleavage domain.

5. The ZFN of claim 4, wherein the ZFN cleaves one or more EPSPS paralogous or orthologous gene sequences.

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 one or more polynucleotides according to claim 6 into the plant cells; and

expressing the one or more ZFNs in the cell, wherein the ZFNs 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 EPSPS paralogous genes in the plant cell are cleaved.

28. A method for stimulating intramolecular homologous recombination in the genome of a plant cell, the method comprising the step of:

introducing a ZFN according to claim 4 into the plant cell in the presence of DNA segment comprising an EPSPS gene and a first sequence that is homologous to a second sequence in the plant cell such that the ZFN cleaves the DNA segment and stimulates intramolecular homologous recombination.

29. The method of claim 28, wherein homologous recombination results in a deletion within the endogenous DNA segment.

30. The method of claim 29, wherein the deleted sequences are selected from the group consisting of sequences encoding all or part of an EPSPS gene and sequence encoding all or part of a selectable marker.

31. The method of claim 29, wherein the deleted DNA is replaced by an exogenous sequence, the method further comprising:

introducing a polynucleotide into the cell, wherein the polynucleotide comprises;

(a) fourth and fifth sequences, wherein the fourth sequence is homologous to non-deleted sequences in proximity to the first sequence and the fifth sequence is homologous to non-deleted sequences in proximity to the second sequence; and

(b) the exogenous sequence.

32. The method of claim 31, wherein the exogenous sequence is a selectable marker.

33. The method of claim 32, wherein the selectable marker is selected from the group consisting of green fluorescent protein (GFP), β

-glucuronidase (GUS), phosphinothricin N-acetyl transferase (PAT, BAR), neomycin phosphotransferase, hygromycin phosphotransferase, β

-lactamase, catechol dioxygenase, α

-amylase, tyrosinase, β

-galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase.

34. The method of claim 31, wherein the exogenous sequence is an EPSPS gene sequence.

35. The method of claim 34, wherein the EPSPS gene sequence comprises a mutation.

36. The method of claim 35, wherein the mutation increases tolerance of a plant to the herbicide glyphosate.

37. A method for deleting an EPSPS gene sequence from the genome of a plant cell, the method comprising:

expressing first and second zinc finger nucleases (ZFNs) according to claim 4 in the cell, wherein the first ZFN cleaves at a first cleavage site in an ESPS gene and the second ZFN cleaves at a second cleavage site in the ESPS gene, wherein the EPSPS gene sequence is located between the first cleavage site and the second cleavage site, wherein cleavage of the first and second cleavage sites results in deletion of the EPSPS gene sequence.

38. The method of claim 37, wherein the EPSPS gene sequence is deleted by non-homologous end joining of first and second cleavage sites.

39. The method of claim 37, wherein the first and second cleavage sites are separated by at least 100 nucleotides.

40. The method of claim 37, wherein the plant cell is a transgenic plant cell comprising an exogenous ESPS sequence.

41. A method for modulating expression of an EPSPS gene, the method comprising:

expressing a ZFP in the cell according to claim 1, wherein said ZFP binds to a target site in the EPSPS gene, thereby modulating expression of the EPSPS gene.

42. The method of claim 41, wherein the ZFP increases transcription of the EPSPS gene.

43. The method of claim 42, wherein the ZFP increases tolerance of a plant to the herbicide glyphosate.

44. The method of claim 41, wherein the ZFP decreases transcription of the EPSPS gene.

45. The method of claim 44, wherein the ZFP decreases tolerance of a plant to the herbicide glyphosate.

15. A donor vector comprising first and second DNA sequences;

wherein the first sequence is homologous to a third sequence and the second sequence is homologous to a fourth sequence; and

wherein the third and fourth sequences are chromosomal EPSPS DNA sequences.

16. The donor vector of claim 15, wherein the near edges of third and fourth sequences are contiguous.

17. The donor vector of claim 15, wherein the near edges of the third and fourth sequences are separated by at least 1 nucleotide pair.

18. The vector of claim 15, wherein the third and fourth sequences are exogenous sequences.

19. The vector of claim 15, wherein the third and fourth sequences are endogenous sequences.

20. The vector of claim 15, further comprising a fifth sequence, wherein the fifth sequence:

(a) is interposed between the first and second sequences; and

(b) is an exogenous nucleic acid sequence.

21. The vector of claim 20, wherein the fifth sequence comprises sequences encoding a selectable marker.

22. The vector of claim 21, wherein the selectable marker is selected from the group consisting of green fluorescent protein (GFP), β

-glucuronidase (GUS), phosphinothricin N-acetyl transferase (PAT, BAR), neomycin phosphotransferase, hygromycin phosphotransferase, β

-lactamase, catechol dioxygenase, α

-amylase, tyrosinase, β

-galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase.

23. The vector of claim 20, wherein the fifth sequence comprises sequences selected from the group consisting of a sequence encoding a protein other than a selectable marker;

one or more transcriptional regulatory sequences;

one or more sequences which enhance or diminish protein targeting;

one or more sequences encoding a portion of a protein;

a small interfering RNA; and

a micro RNA.

24. The vector of claim 23, wherein the fifth sequence comprises a sequence encoding a mutant EPSPS chromosomal sequence that increases tolerance of a plant to the herbicide glyphosate.

25. A method for introducing an exogenous nucleic acid sequence into the genome of a plant cell, the method comprising the step of:

expressing one or more zinc finger nucleases (ZFN) in the cell, in the presence of a donor vector according to claim 15;

wherein the ZFN cleaves an EPSPS gene in chromosomal DNA within 3 kilobase pairs of either of the third or fourth sequences;

such that cleavage of the chromosomal DNA in step (b) stimulates incorporation of the donor vector into the genome by homologous recombination.

26. A method for expressing the product of an exogenous nucleic acid sequence in a plant cell, the method comprising the steps of:

introducing the exogenous sequence according to the method of claim 25 such that the product of the exogenous sequence is expressed in the plant cell.

27. A transgenic plant cell or transgenic plant obtained according to the method of claim 25.