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 LRGSPISRARPLNPHP (SEQ ID NO:272); LRGSISRARPLNPHP (SEQ ID NO:273); LRGSPSRARPLNPHP (SEQ ID NO:274); LRGSSRARPLNPHP (SEQ ID NO:275); LRGSYAPMPPLALASP (SEQ ID NO:276); LRGSAPMPPLALASP (SEQ ID NO:277); LRGSPMPPLALASP (SEQ ID NO:278); or LRGSMPPLALASP (SEQ ID NO:279).
2. The fusion protein of claim 1 , wherein the linker comprises LRGSISRARPLNPHP (SEQ ID NO:273).
3. The fusion protein of claim 1 , wherein the linker comprises LRGSYAPMPPLALASP (SEQ ID NO:276).
4. The fusion protein of claim 1 , wherein the DNA-binding domain is a zinc finger protein.
5. The fusion protein of claim 1 , wherein the N-terminal residues QLVKS (residues 158 to 162 of SEQ ID NO:1) of the FokI cleavage domain are deleted.
6. A dimer comprising two fusion proteins according to claim 1 .
7. The dimer of claim 6 , wherein the dimer is a homodimer or heterodimer.
8. A polynucleotide encoding at least one fusion protein according to claim 1 .
9. A cell comprising a fusion protein according to claim 1 .
10. A cell comprising a polynucleotide according to claim 8 .
11. 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 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 .
12. The method of claim 11 , wherein both nucleases comprise fusions proteins according to claim 1 .
13. The method of claim 11 , wherein the target sites for the zinc finger nucleases are 3 to 20 base pairs apart.
14. The method of claim 11 , 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.
15. 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.
16. A kit for producing a nuclease, the kit comprising a polynucleotide according to claim 8 contained in one or more containers, optional hardware, and instructions for use of the kit.
17. The kit of claim 15 , further comprising a donor polynucleotide.