Site-specific enzymes and methods of use
The present invention provides polypeptides related to Ralstonia proteins, nucleic acids encoding the same, compositions comprising the same, kits comprising the same, non-human transgenic animals comprising the same, and methods of using the same.
1. A fusion protein comprising
(a) at least one amino acid sequence having the sequence of
LSTEQVVAIASX 1 X 2 GGKQALEAVKAQLLVLRAAPYE (SEQ ID NO: 1), wherein X 1 X 2 comprises a repeat variable diresidue (RVD), and wherein the RVD consists of SI, SN, SH, NP, NH, NT, NK, ND, HN, HY, HD, HH, RN, RS, NG or GS and
(b) an effector domain,
wherein the effector domain comprises a nuclease, a nickase, a transcriptional activator, a transcriptional repressor, a methyltransferase, a deacetylase or any functional fragment thereof and
wherein the effector domain is neither isolated nor derived from a Ralstonia TALEN.
2. The fusion protein of claim 1 , further comprising a second amino acid sequence having the sequence of
(SEQ ID NO: 1)
LSTEQVVAIASX 1 X 2 GGKQALEAVKAQLLVLRAAPYE,
wherein X 1 X 2 comprises a repeat variable diresidue (RVD), and wherein the RVD consists of SI, SN, SH, NP, NH, NT, NK, ND, HN, HY, HD, HH, RN, RS, NG or GS.
3. The fusion protein of claim 1 or 2 , wherein the at least one amino acid sequence of (a) or the second amino acid sequence comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO: 19.
4. The fusion protein of any one of claims 1 or 2 wherein the effector domain comprises at least one nuclease.
5. The fusion protein of any one of claims 1 or 2 wherein the nuclease comprises an endonuclease.
6. The fusion protein of claim 5 , wherein the endonuclease comprises a Fok I endonuclease.
7. The fusion protein of claim 5 , wherein the endonuclease comprises an I-SceI endonuclease.
8. The fusion protein of any one of claims 1 or 2 wherein the effector domain comprises a zinc-finger nuclease.