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 method for inserting a sequence into an endogenous inositol polyphosphate 2-kinase (IPP2K) gene in a maize cell, the method comprising:
expressing, in the maize cell, a pair of zinc finger nucleases that bind to and cleave the plant cellular chromatin in a region of interest, wherein the region of interest is in an IPP2K gene and further wherein at least one zinc finger nuclease comprises a zinc finger protein having 4, 5 or 6 zinc finger domains designated and ordered F1 to F4, F1 to F5 or F1 to F6, each zinc finger domain comprising a recognition helix region and further wherein the zinc finger protein is selected from the group consisting of a protein comprising the recognition helix regions of the sequence and order as shown in a single row of Table 8; and
introducing the sequence into the maize cell such that the sequence is inserted into at least two alleles of the same IPP2K gene in the region of interest.
2. The method of claim 1 , wherein a single-copy of the sequence is inserted into the region of interest.
3. The method of claim 2 , wherein the sequence is also randomly inserted into the cellular chromatin.
4. The method of claim 1 , wherein insertion of the sequence inactivates the IPP2K gene.
5. The method of claim 1 , wherein the zinc finger nucleases are transiently expressed in the maize cell.
6. The method of claim 1 , wherein the sequences encoding the zinc finger nucleases are stably integrated into the genome of the maize cell.
7. The method of claim 1 , wherein the sequence is homologous and non-identical to a second sequence in the region of interest.
8. The method of claim 1 , wherein at least one zinc finger nuclease comprises a non-canonical zinc finger having a helical portion involved in DNA binding and further wherein the at least one non-canonical 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.
9. The method of claim 1 , wherein at least one zinc finger nuclease comprises a non-canonical zinc finger having a helical portion involved in DNA binding and further wherein the at least one non-canonical zinc finger comprises the sequence HX 1 X 2 RCX L (SEQ ID NO:2), where X 1 , X 2 and X L can be any amino acid.
10. A maize cell comprising an inserted sequence in an endogenous IPP2K gene, wherein the insertion is achieved by the method of claim 1 .
11. A maize plant, maize plant part, or maize seed, comprising the cell of claim of claim 10 .