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 fusion protein comprising:
(a) a cleavage half domain;
(b) 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 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 wherein
(i) the zinc finger protein is engineered to bind to a target sequence;
(ii) (X D ) 1-10 comprises an amino acid sequence selected from the group consisting of a G residue as the N-terminal residue of (X D ) 1-10 ; QKP; and QLV;
(iii) (X C ) 3-5 comprises N-terminal T, L, A, Q, KE, KK, KQ, KI, KRI, SEGG, SSSN, IKR, IRT, ISS or IRR residues, and
(iv) if (X C ) 3-5 consists of the amino acid residues TKI then the N-terminal residues of (X D ) 1-10 comprise GG, GL or GT; and
(c) a ZC linker interposed between the cleavage half-domain and the zinc finger protein, wherein the ZC linker has the sequence GLRGS (SEQ ID NO:4) or GGLRGS (SEQ ID NO:5).
2. The fusion protein of claim 1 , wherein the region of the zinc finger protein corresponding to His-(X C ) 3-5 -Cys-(X D ) 1-10 consists of one of the amino acid sequences shown in Tables 1, 2, 3, or 4.
3. The fusion protein of claim 1 , wherein X D comprises the sequence QLV or QKP.
4. The fusion protein of claim 3 , wherein the sequence QLV or QKP are the 3 C-terminal amino acid residues of the zinc finger.
5. The fusion protein of claim 1 , wherein (X D ) 1-10 comprises 1, 2 or 3 Gly (G) residues.
6. A fusion protein comprising 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 .
7. The fusion protein of claim 6 , wherein the zinc finger protein comprises 3, 4, 5 or 6 zinc fingers.
8. The fusion protein of claim 6 , wherein finger 2 comprises the CCHC zinc finger.
9. The fusion protein of claim 6 , wherein the C-terminal zinc finger comprises the CCHC finger.
10. The fusion protein of claim 6 , wherein at least two zinc fingers comprise the CCHC zinc finger.
11. The fusion protein of claim 6 , 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.
12. A polynucleotide encoding a fusion protein according to claim 1 .
13. A plant cell comprising a fusion protein according to claim 1 .
14. The plant cell of claim 13 , wherein the cell is present in a seed.
15. The plant cell of claim 14 , wherein seed is a corn seed.
16. The plant cell of claim 13 , wherein IPP2-K is partially or fully inactivated.
17. The plant cell of claim 14 , wherein the levels of phytic acid in the seed are reduced.
18. The plant cell of claim 13 , wherein metabolically available levels of phosphorous in the cell are increased.