Modified DNA-binding proteins and uses thereof

Patent metadata

Field	Value
Publication number	US-9458205-B2
Application number	US-201213679684-A
Country	US
Kind code	B2
Filing date	Nov 16, 2012
Priority date	Nov 16, 2011
Publication date	Oct 4, 2016
Grant date	Oct 4, 2016

Abstract

Disclosed herein are enhanced polypeptides, polynucleotides encoding these polypeptides, 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.

First claim

What is claimed is:

1. An isolated, non-naturally occurring transcription activator-like effector (TALE) DNA-binding polypeptide comprising a plurality of TALE-repeat units, each TALE-repeat unit comprising a repeat variable di-residue (RVD) region, wherein the TALE DNA-binding polypeptide comprises at least 3 non-canonical RVDs, and wherein the non-canonical RVDs are CI or KI for recognition of an adenine; AD, KD, or RD for recognition of a cytosine; AN, CN, GN, FN, AK or CK for recognition of guanine; and KG, MG, QG, RG, AA, QA or VA for recognition of thymine.

2. The TALE DNA-binding polypeptide of claim 1 , wherein the amino acid at position 11 in at least one of the repeat units has been altered as compared to a naturally occurring TALE repeat unit.

3. The TALE DNA-binding polypeptide of claim 2 , wherein the amino acid at position 11 is altered to an amino acid selected from the group consisting of an Alanine, Cysteine, Glycine, Histidine, Lysine, Methionine, Asparagine, Glutamine and Arginine.

4. A kit comprising a TALE DNA-binding polypeptide according to claim 1 .

5. A fusion protein comprising the TALE DNA-binding polypeptide of claim 1 and a functional domain.

6. The fusion protein of claim 5 , wherein the functional domain is selected from the group consisting of a transcriptional activator, a transcriptional repressor, a methyltransferase and a nuclease cleavage domain.

7. The fusion protein according to claim 5 , wherein the nuclease cleavage domain comprises a FokI cleavage domain.

8. A dimer comprising the fusion protein of claim 7 and a protein comprising a TALE DNA binding domain polypeptide linked to an inactive FokI catalytic domain.

9. A polynucleotide encoding the TALE DNA-binding polypeptide of claim 1 .

10. A polynucleotide encoding the fusion protein of claim 6 .

11. An isolated host cell comprising the TALE DNA-binding polypeptide of claim 1 .

12. The isolated host cell of claim 11 , wherein the cell is a eukaryotic cell.

13. The isolated host cell of claim 12 , wherein the cell is a mammalian or plant cell.

14. The isolated host cell of claim 13 , wherein the mammalian cell is a stein cell.

15. A method of modulating expression of an endogenous gene in a cell, the method comprising:
introducing into the cell a fusion protein according to claim 6 , wherein the fusion protein binds to a target site in the endogenous gene, and wherein expression of the endogenous gene is modulated by the fusion protein.

16. The method of claim 15 , wherein the modulation is selected from the group consisting of gene activation, gene repression and gene inactivation.

17. The method of claim 16 , wherein the fusion protein comprises a cleavage domain or cleavage half-domain and the endogenous gene is inactivated by cleavage.

18. The method of claim 15 , wherein the fusion protein is introduced as a polynucleotide encoding the fusion protein.

19. A method of modifying a region of interest in a genome of a cell, the method comprising:
introducing into the cell at least one fusion protein according to claim 7 , wherein the fusion protein binds to a target site in the genome of the cell and cleaves the genome in the region of interest.

20. The method of claim 19 , wherein the modifying comprises introducing a deletion in the region of interest.

21. The method of claim 19 , 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.

22. The method of claim 19 , wherein the cell is a eukaryotic cell selected from the group consisting of a plant cell, an animal cell, a fish cell and a yeast cell.

23. The method of claim 19 , wherein the fusion protein is introduced as a polynucleotide encoding the fusion protein.

24. A method of making a single-stranded break in a double-stranded DNA target sequence, the method comprising:
binding the dimer according to claim 8 with the double-stranded DNA target sequence.

25. A method of cleaving a double-stranded DNA target, the method comprising:
binding first and second dimers according to claim 8 with the double-stranded DNA target, such that the first and second dimers make first and second single-stranded breaks on complementary strands of the double-stranded DNA target, wherein the first and second single-stranded breaks result in separation of the double-stranded DNA into two fragments, thereby cleaving the double-stranded DNA target.

Assignees

• Sangamo Biosciences Inc

Inventors

• Gregory Philip D
• Miller Jeffrey C
• Paschon David
• Rebar Edward J
• Tan Siyuan
• Urnov Fyodor
• Zhang Lei

Classifications

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C07K14/195 Primary

  from bacteria · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N5/0602

  Vertebrate cells · CPC title

• C07K2319/80

  containing a DNA binding domain, e.g. Lacl or Tet-repressor · CPC title