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 with alternative configurations. Also described are methods of making and using compositions comprising these linkers.
1. A dimer comprising first and second fusion molecules, the first fusion molecule comprising a DNA binding domain that binds to a first target site and a first wild-type or engineered cleavage domain and the second fusion molecule comprising a DNA-binding domain that binds to a second target site and a second wild-type or engineered cleavage domain, wherein the first and second cleavage domains dimerize when the DNA-binding domains are bound to the first and second target sites separated by 6 to 7 base pairs, wherein the first fusion molecule comprises an amino acid linker between the DNA-binding domain and the first cleavage domain, wherein the linker is selected from the group consisting of:
(a) when the first and second target sites are separated by 6 base pairs, the linker is selected from the group consisting of: SGSLRGVDPMWH (SEQ ID NO:78); SGRSPEMDWC (SEQ ID NO:62); GASLGPPWCP (SEQ ID NO:80); SGLPMGSYGS (SEQ ID NO:81); SGQSPGDVGF (SEQ ID NO:71); SGAIYARPIE (SEQ ID NO:82); SGAQGSTLDF (SEQ ID NO:83); or SGVKRDSEII (SEQ ID NO:84);
(b) when the first and second target sites are separated by 7 base pairs the linker is selected from the group consisting of: SQATPTLYYTPL (SEQ ID NO:88); SGQPMFSWSD (SEQ ID NO: 109).
2. A dimer according to claim 1 , wherein the DNA-binding domains comprises a zinc finger protein, a TAL-effector domain or a single guide RNA (sgRNA).
3. A dimer according to claim 1 , wherein the linker extends between the N-terminal of the DNA-binding domain and C-terminal of the cleavage domain.
4. The dimer according to claim 1 wherein the linker comprises SGQSPGDVGF (SEQ ID NO:71) or SGVKRDSEII (SEQ ID NO:84).
5. The dimer according to claim 1 , wherein the DNA-binding domains bind to opposite or the same strand of DNA in a double-stranded target DNA.
6. The dimer according to claim 1 , wherein the dimer makes a double- or single-stranded cut in a target double-stranded DNA.
7. A polynucleotide encoding the dimer of claim 1 .
8. An isolated cell comprising one or more dimers according to claim 1 .
9. A method of modifying cellular chromatin in a cell, the method comprising: cleaving the cellular chromatin with a dimer according to claim 1 such that the cellular chromatin is modified, wherein the dimer is introduced into the cell using one or more polynucleotides.
10. The method according to claim 9 , wherein the modification comprises introducing an insertion and/or deletion into the cellular chromatin.
11. The method according to claim 9 , wherein the modification comprises integration of a donor sequence.
12. A kit comprising a dimer according to claim 1 .