Engineered cleavage half-domains

• US 8,623,618 B2
• Filed: 12/30/2011
• Issued: 01/07/2014
• Est. Priority Date: 02/08/2010
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Doyon, Yannick, Miller, Jeffrey C.
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US13/341,537
• Publication Number:
  US 20120142062A1
• Time in Patent Office: 739 Days
• Field of Search: None
• US Class Current: 435/91.53
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C07K 7/06 having 5 to 11 amino acids
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/10 Transferases (2.) ribonucleases C12N9/22
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/21004 Type II site-specific deoxyribonuclease (3.1.21.4)

Abstract

Disclosed herein are engineered cleavage half-domains; fusion polypeptides comprising these engineered cleavage half-domains; polynucleotides encoding the engineered cleavage half-domains and fusion proteins; and cells comprising said polynucleotides and/or fusion proteins. Also described are methods of using these polypeptides and polynucleotides, for example for targeted cleavage of a genomic sequence.

Claims

1. A TALE-nuclease (TALEN) fusion polypeptide comprising:

(i) a TALE effector DNA-binding domain; and

(ii) a polypeptide comprising an engineered FokI cleavage half-domain, wherein the engineered cleavage half-domain comprises a mutation selected from the group consisting of;

substitution mutations at amino acid residues 486, 499 and 496;

substitution mutations at amino acid residues 487 and 496;

substitution mutations at amino acid residues 487, 499, and 496;

substitution mutations at amino acid residues 483 and 537;

substitution mutations at amino acid residues 490 and 537;

substitution mutations at amino acid residues 490, 537 and 538;

substitution mutations at amino acid residues 483, 496 and 537;

substitution mutations at amino acid positions 487, 496 and 537;

substitution mutations at amino acid residues 487, 499 and 496; and

substitution mutations at amino acid residues 483, 537 and 538,

wherein the amino acid residues are numbered relative to full length wild-type FokI as shown in SEQ ID NO;

57.

2. The fusion polypeptide of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 486, 499 and 496 and further wherein the wild-type Gln (Q) residue at position 486 is replaced with a Glu (E) residue, the wild-type Iso (I) residue at position 499 is replaced with a Leu (L) residue and the wild-type Asn (N) residue at position 496 is replaced with an Asp (D) or a Glu (E) residue.

3. The fusion polypeptide of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 490, 538 and 537 and further wherein the wild-type Glu (E) residue at position 490 is replaced with a Lys (K) residue, the wild-type Iso (I) residue at position 538 is replaced with a Lys (K) residue, and the wild-type His (H) residue at position 537 is replaced with a Lys (K) residue or a Arg (R) residue.

4. The fusion polypeptide of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 490 and 537 and further wherein the wild-type Glu (E) residue at position 490 is replaced with a Lys (K) residue and the wild-type His (H) residue at position 537 is replaced with a Lys (K) residue or a Arg (R) residue.

5. The fusion polypeptide of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 487 and 496 and further wherein the wild-type Arg (R) residue at position 487 is replaced with an Asp (D) residue- and the wild-type Asn (N) residue at position 496 is replaced with an Asp (D) residue.

6. The fusion polypeptide of claim 5, further wherein the wild-type Ile (I) residue at position 499 is replaced with an Ala (A).

7. The fusion polypeptide of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 483 and 537 and further wherein the wild-type Asp (D) residue at position 483 is replaced with an Arg (R) residue and the wild-type His (H) residue at position 537 is replaced with an Arg (R) residue.

8. The fusion polypeptide of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 487, 496 and 537 and further wherein the wild-type Arg (R) residue at position 487 is replaced with an Asp (D) residue, the wild-type Asn (N) residue at position 496 is replaced with an Asp (D) residue and the wild-type His (H) residue at position 537 is replaced with an Arg (R) residue.

9. The fusion polypeptide of claim 1, wherein the engineered cleavage half-domain comprises substitution mutations at amino acid residues 483, 496 and 537 and further wherein the wild-type Asp (D) residue at position 483 is replaced with an Arg (R) residue, the wild-type Asn (N) residue at position 496 is replaced with an Asp (D) residue and the wild-type His (H) residue at position 537 is replaced with an Arg (R) residue.

10. The fusion polypeptide of claim 1, further comprising an additional amino acid substitution at one or more of positions 418, 432, 441, 481, 483, 486, 487, 490, 496, 499, 523, 527, 537, 538 and 559.

11. A heterodimer comprising a first fusion polypeptide of claim 1 and a second TALEN fusion polypeptide.

12. A polynucleotide encoding the fusion polypeptide of claim 1.

13. An isolated cell comprising the polypeptide of claim 1.

14. An isolated cell comprising the polynucleotide of claim 11.

15. A method for cleaving genomic cellular chromatin in a region of interest, the method comprising:

(a) expressing in a cell a first TALEN fusion polypeptide according to claim 1 in a cell, wherein the TALE-effector DNA-binding domain binds to a first target site in the region of interest;

(d) expressing a second fusion protein in the cell, the second TALEN fusion protein comprising a second TALE effector DNA-binding domain engineered to bind to the second target site and a second engineered cleavage half-domain,

wherein the first and second TALENs form a heterodimer that cleaves the cellular chromatin in the region of interest.

16. The method of claim 15, wherein cleavage occurs between the first and second target sites.

17. The method of claim 15, further comprising contacting the cell with a donor polynucleotide;

wherein cleavage of the cellular chromatin in the region of interest results in integration of the donor polynucleotide into the region of interest.

18. A method of cleaving at least two target sites in genomic cellular chromatin, the method comprising:

cleaving at least first and second target sites in genomic cellular chromatin, wherein each target site is cleaved using a pair of TALENs according to claim 1.