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 generating a genetically modified non-human organism, the method comprising:
(a) introducing an mRNA encoding a fusion protein into a gonad, ova or embryo cell of the organism, the fusion protein comprising
(i) 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 no more than residues N+1 to N+137 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 of no more than residues C-20 to residues C+63 of the TALE protein C-terminal domain, and
(ii) a cleavage domain or cleavage half-domain; wherein the fusion protein makes a double-stranded break in an endogenous gene such that the gonad, ova or embryo cell is genetically modified and
(b) allowing the gonad, ova or embryo cell to mature into an adult organism, thereby generating a genetically modified non-human organism.
2. The method of claim 1 , wherein the organism is a plant.
3. The method of claim 1 , wherein the organism is a worm or a mammal.
4. The method of claim 1 , wherein the organism is a mammal.
5. The method of claim 1 , wherein genetic modification occurs via non-homologous end joining (NHEJ).
6. The method of claim 1 , wherein the genetic modification comprises a deletion.
7. The method of claim 1 , wherein the genetic modification comprises an insertion.
8. The method of claim 7 , wherein the insertion comprises an exogenous nucleic acid.
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 comprises a TALE repeat domain.
12. The method of claim 1 , wherein the cleavage domain or cleavage half-domain is from a TypeIIS endonuclease.