Compositions for linking DNA-binding domains and cleavage domains
Disclosed herein are compositions for linking DNA binding domains and cleavage domains (or cleavage half-domains) to form non-naturally occurring nucleases. Also described are methods of making and using compositions comprising these linkers.
1. A fusion protein comprising
a DNA-binding domain having an N-terminus and a C-terminus, wherein the DNA-binding domain binds to a nucleotide target site;
a FokI cleavage domain having an N-terminus and a C-terminus; and
a linker between the C-terminus of the DNA-binding domain and the N-terminus of the cleavage domain, wherein the linker comprises a sequence selected from the group consisting of PKPAN (SEQ ID NO:31), RARPLN (SEQ ID NO:32); PMPPLA (SEQ ID NO:33) or PPPRP (SEQ ID NO:34).
2. The fusion protein of claim 1 , wherein the linker further comprises a ZC sequence (SEQ ID NO:35).
3. The fusion protein of claim 2 , wherein the ZC sequence is at the N-terminus of the linker.
4. The fusion protein of claim 1 , wherein the DNA-binding domain is a zinc finger protein.
5. A dimer comprising two fusion proteins according to claim 1 .
6. The dimer of claim 5 , wherein the dimer is a homodimer or heterodimer.
7. A polynucleotide encoding at least one fusion protein according to claim 1 .
8. A cell comprising a fusion protein according to claim 1 .
9. A cell comprising a polynucleotide according to claim 7 .
10. A method for targeted cleavage of cellular chromatin in a region of interest in a cell, the method comprising:
expressing a pair of nucleases in the cell under conditions such that cellular chromatin is cleaved at a the region of interest, wherein the nucleases bind to target sites in the region of interest and further wherein at least one nuclease comprises a fusion protein according to claim 1 .
11. The method of claim 10 , wherein both nucleases comprise fusions proteins according to claim 1 .
12. The method of claim 10 , wherein the target sites for the zinc finger nucleases are 3 to 20 base pairs apart.
13. The method of claim 10 , further comprising the step of introducing a donor polynucleotide into the cell, wherein all or part of the donor polynucleotide is incorporated into the region of interest following cleavage.
14. A kit for producing a nuclease, the kit comprising a fusion protein according to claim 1 contained in one or more containers, optional hardware, and instructions for use of the kit.
15. A kit for producing a nuclease, the kit comprising a polynucleotide according to claim 7 contained in one or more containers, optional hardware, and instructions for use of the kit.
16. The kit of claim 14 , further comprising a donor polynucleotide.