DNA-binding proteins and uses thereof
Disclosed herein are polypeptides, polynucleotides encoding, cells and organisms comprising novel DNA-binding domains, including TALE DNA-binding domains. Also disclosed are methods of using these novel DNA-binding domains for modulation of gene expression and/or genomic editing of endogenous cellular sequences.
1. A method of modifying an endogenous gene in a region of interest in a cell, the method comprising:
introducing into the cell a polynucleotide encoding a fusion protein, the fusion protein comprising
(a) an isolated, non-naturally occurring DNA-binding polypeptide comprising:
two or more TALE-repeat units, the TALE repeat units comprising a repeat variable di-residue (RVD);
an N-cap polypeptide, wherein the N-cap comprises residues N+1 to N+121 of a TALE protein; and
a C-cap polypeptide, wherein the C-cap polypeptide comprises a fragment of a full length C-terminus region of a TALE protein, the C-cap comprising residues C-1 to C-20 of the TALE protein, and
(b) a functional domain,
wherein the fusion protein binds to a target site in the endogenous gene and further wherein the endogenous gene is modified.
2. The method of claim 1 , wherein the functional domain comprises a transcriptional activation domain and the modifying comprises gene activation.
3. The method of claim 1 , wherein the functional domain comprises a transcriptional repression domain and the modifying comprises gene repression or inactivation.
4. The method of claim 1 , wherein the functional domain comprises a cleavage domain or cleavage half-domain and the endogenous gene is inactivated by cleavage.
5. The method of claim 4 , wherein inactivation occurs via non-homologous end joining (NHEJ).
6. The method of claim 4 , wherein the modifying comprises introducing a deletion in the region of interest.
7. The method of claim 4 , wherein the modifying comprises introducing an exogenous nucleic acid into the region of interest, the method further comprising introducing the exogenous nucleic acid into the cell, wherein the exogenous nucleic acid is integrated into the region of interest by homologous recombination.
8. The method of claim 1 , wherein the cell is a eukaryotic cell selected from the selected from the group consisting of a plant cell, an animal cell, a fish cell and a yeast cell.
9. The method of claim 1 , wherein at least one TALE-repeat unit comprises an atypical repeat variable di-residue (RVD).
10. The method of claim 9 , wherein the protein comprises an atypical RVD as shown in Tables 27A or 27B.
11. The method of claim 1 , wherein the C-cap polypeptide is less than approximately 230 amino acids in length.
12. The method of claim 1 , wherein the C-cap comprises a TALE repeat domain.
13. The method of claim 4 , wherein the cleavage domain or cleavage half-domain is from a Type IIS endonuclease.