Optimized non-canonical zinc finger proteins
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.
1. A non-naturally occurring zinc finger protein comprising a non-canonical (non-C2H2) zinc finger, wherein the non-canonical zinc finger has a non-naturally occurring helical portion involved in DNA binding and 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 and His-(X C ) 3-5 -Cys-(X D ) 1-10 comprises a sequence as shown in any of SEQ ID NOs:14-31, 33-80, 83-87 and 89-93 and further wherein (X B ) 6-12 of (X B ) 12 is the non-naturally occurring helical portion involved in DNA binding such that the non-naturally occurring zinc finger protein is engineered to bind to a target sequence.
2. 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 .
3. The zinc finger protein of claim 2 , wherein the zinc finger protein comprises 3, 4, 5 or 6 zinc fingers.
4. The zinc finger protein of claim 2 , wherein finger 2 comprises the CCHC zinc finger.
5. The zinc finger protein of claim 2 , wherein the C-terminal zinc finger comprises the CCHC finger.
6. The zinc finger protein of claim 2 , wherein at least two zinc fingers comprise the CCHC zinc finger.
7. The zinc finger protein of claim 2 , 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.
8. A fusion protein comprising a zinc finger protein of claim 1 and one or more functional domains.
9. The fusion protein of claim 8 , wherein the functional domain is a cleavage domain.
10. A polynucleotide encoding a zinc finger protein according to claim 1 .
11. 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 9 ;
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.
12. 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 9 , 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.
13. The method of claim 11 , 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.
14. The method of claim 11 , further comprising introducing an exogenous polynucleotide into the host cell.
15. The method of claim 14 , wherein the exogenous polynucleotide comprises sequences homologous to the host target locus.
16. The method of claim 11 , wherein the plant is selected from the group consisting of a monocotyledon, a dicotyledon, gymnosperms and eukaryotic algae.
17. The method of claim 11 , wherein the plant is selected from the group consisting of maize, rice, wheat, cotton, rice, potato, soybean, tomato, tobacco, members of the Brassica family, and Arabidopsis .
18. The method of claim 11 , wherein the plant is a tree.
19. The method of claim 11 , wherein the target sequences are in an IPP2-K gene.
20. A method for reducing the level of phytic acid in seeds, the method comprising inactivating or altering an IPP2-K gene according to claim 7 .
21. A method for making phosphorous more metabolically available in seed, the method comprising inactivating or altering an IPP2-K gene according to claim 7 .
22. A plant cell comprising a zinc finger protein according to claim 1 .
23. The plant cell of claim 22 , wherein the cell is present in a seed.
24. The plant cell of claim 23 , wherein seed is a corn seed.
25. The plant cell of claim 22 , wherein IPP2-K is partially or fully inactivated.
26. The plant cell of claim 23 , wherein the levels of phytic acid in the seed are reduced.
27. The plant cell of claim 22 , wherein metabolically available levels of phosphorous in the cell are increased.