Optimized non-canonical zinc finger proteins

• US 20080182332A1
• Filed: 12/13/2007
• Published: 07/31/2008
• Est. Priority Date: 12/14/2006
• Status: Active Grant

• Current Assignee: Dow Agrosciences LLC (Corteva, Inc.), Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Urnov, Fyodor, Petolino, Joseph F., Miller, Jeffrey, Cai, Qihua C., Baker, Lisa W., Blue, Ryan C., Arnold, Nicole L., Garrison, Robbi J., Mitchell, Jon C., Worden, Sarah E., Shukla, Vipula K.
• Granted Patent: US 9,187,758 B2
• Time in Patent Office: Days
• US Class Current: 435/468
• 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

Abstract

Disclosed herein are zinc fingers comprising CCHC zinc coordinating residues. Also described are zinc finger proteins and fusion proteins comprising these CCHC zinc fingers as well as polynucleotides encoding these proteins. Methods of using these proteins for gene editing and gene regulation are also described.

Claims

1. A zinc finger protein comprising a non-canonical (non-C₂H₂) zinc finger, wherein the non-canonical zinc finger has a helical portion involved in DNA binding and wherein the zinc-coordinating region of the helical portion comprises the amino acid sequence HX₁X₂RCX_L (SEQ ID NO:

2); and

wherein the zinc finger protein is engineered to bind to a target sequence.

2. The zinc finger protein of claim 1, wherein X₁ is A and X₂ is Q.

3. The zinc finger protein of claim 1, wherein X₁ is K and X₂ is E.

4. The zinc finger protein of claim 1, wherein X₁ is T and X₂ is R.

5. The zinc finger protein of claim 1, wherein X_L is G.

6. A zinc finger protein of claim 1, wherein at least one zinc finger comprises the sequence Cys-(X^A)_2-4-Cys-(X^B)₁₂-His-(X^C)_3-5-Cys(X^D)_1-10 (SEQ ID NO:

3), where X^A, X^B, X^C and X^D can be any amino acid.

7. The zinc finger protein of claim 1, comprising any of the sequences shown in any of Tables 1, 2, 3 or 4.

8. The zinc finger protein of claim 6, wherein X^D comprises the sequence QLV or QKP.

9. The zinc finger protein of claim 8, wherein the sequence QLV or QKP are the 3 C-terminal amino acid residues of the zinc finger.

10. The zinc finger protein of claim 6, wherein X^D comprises 1, 2 or 3 Gly (G) residues.

11. A zinc finger protein comprising a plurality of zinc fingers, wherein at least one of the zinc fingers comprises a CCHC zinc finger according to claim 1.

12. The zinc finger protein of claim 11, wherein the zinc finger protein comprises 3, 4, 5 or 6 zinc fingers.

13. The zinc finger protein of claim 11, wherein finger 2 comprises the CCHC zinc finger.

14. The zinc finger protein of claim 11, wherein the C-terminal zinc finger comprises the CCHC finger.

15. The zinc finger protein of claim 11, wherein at least two zinc fingers comprise the CCHC zinc finger.

16. The zinc finger protein of claim 11, wherein the zinc finger protein comprises any of the sequences shown in Table 8 and is engineered to bind to a target sequence in an IPP2-K gene.

17. A fusion protein comprising a zinc finger protein of claim 1 and one or more functional domains.

18. A fusion protein comprising:

(a) a cleavage half-domain,

(b) the zinc finger protein of claim 1, and

(c) a ZC linker interposed between the cleavage half-domain and the zinc finger protein.

19. The fusion protein of claim 18, wherein the length of the ZC linker is 5 amino acids.

20. The fusion protein of claim 19, wherein the amino acid sequence of the ZC linker is GLRGS (SEQ ID NO:

4).

21. The fusion protein of claim 18, wherein the length of the ZC linker is 6 amino acids.

22. The fusion protein of claim 21, wherein the amino acid sequence of the ZC linker is GGLRGS (SEQ ID NO:

5).

23. A polynucleotide encoding a zinc finger protein according to claim 1.

24. A method for targeted cleavage of cellular chromatin in a plant cell, the method comprising expressing, in the cell, a pair of fusion proteins according to claim 18;

wherein;

(a) the target sequences of the fusion proteins are within ten nucleotides of each other; and

(b) the fusion proteins dimerize and cleave DNA located between the target sequences.

25. A method of targeted genetic recombination in a host plant cell, the method comprising:

(a) expressing, in the host cell, a pair of fusion proteins according to claim 18, wherein the target sequences of the fusion proteins are present in a chosen host target locus; and

(b) identifying a recombinant host cell which exhibits a sequence alteration in the host target locus.

26. The method of claim 24, wherein the sequence alteration is a mutation selected from the group consisting of a deletion of genetic material, an insertion of genetic material, a substitution of genetic material and any combination thereof.

27. The method of claim 24, further comprising introducing an exogenous polynucleotide into the host cell.

28. The method of claim 27, wherein the exogenous polynucleotide comprises sequences homologous to the host target locus.

29. The method of claim 24, wherein the plant is selected from the group consisting of a monocotyledon, a dicotyledon, gymnosperms and eukaryotic algae.

30. The method of claim 29, wherein the plant is selected from the group consisting of maize, rice, wheat, potato, soybean, tomato, tobacco, members of the Brassica family, and Arabidopsis.

31. The method of claim 24, wherein the plant is a tree.

32. The method of claim 24, wherein the target sequences are in an IPP2K gene.

33. A method for reducing the level of phytic acid in seeds, the method comprising inactivating or altering an IPP2-K gene according to claim 32.

34. A method for making phosphorous more metabolically available in seed, the method comprising inactivating or altering an IPP2-K gene according to claim 32.

35. A plant cell comprising a zinc finger protein according to claim 1.

36. The plant cell of claim 35, wherein the cell is a seed.

37. The plant cell of claim 36, wherein seed is a corn seed.

38. The plant cell of claim 35, wherein IPP2-K is partially or fully inactivated.

39. The plant cell of claim 38, wherein the levels of phytic acid in the seed are reduced.

40. The plant cell of claim 35, wherein metabolically available levels of phosphorous in the cell are increased.