Engineered cleavage half-domains

• US 20110201055A1
• Filed: 02/07/2011
• Published: 08/18/2011
• 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.
• Granted Patent: US 8,962,281 B2
• Time in Patent Office: Days
• US Class Current: 435/91.1
• 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 polypeptide comprising an engineered FokI cleavage half-domain, wherein the engineered cleavage half-domain comprises a mutation selected from the group consisting of:

mutations in amino acid residues 486, 499 and 496;

mutations in amino acid residues 487 and 496;

mutations in amino acid residues 487, 499, and 496;

mutations in amino acid residues 483 and 537;

mutations in amino acid residues 490 and 537;

mutations in amino acid residues 490, 537 and 538;

mutations in amino acid residues 483 and 538;

mutations in amino acid residues 487, 499 and 496; and

mutations in amino acid residues 483, 538 and 537, wherein the amino acid residues are numbered relative to full length wild-type FokI.

2. The polypeptide of claim 1, wherein the engineered cleavage half-domain comprises a polypeptide in which 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 polypeptide of claim 1, wherein the engineered cleavage half-domain comprises a polypeptide in which 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 polypeptide of claim 1, wherein the engineered cleavage half-domain comprises a polypeptide in which 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 polypeptide of claim 1, wherein the engineered cleavage half-domain comprises a polypeptide in which 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 polypeptide of claim 5, further wherein the wild-type Ile (I) residue at position 499 is replaced with an Ala (A)

7. The polypeptide of claim 1, wherein the engineered cleavage half-domain comprises a polypeptide in which the wild-type Asp (D) residue at position 483 is replaced with an Arg (R) residue and the polypeptide comprises at least one additional mutation selected from the group consisting of:

(i) a mutation in which the wild-type Ile (I) residue at position 538 is replaced with a Val (V) residue;

(ii) a mutation in which the wild-type His (H) residue at position 537 is replaced with an Arg (R) residue; and

(ii) combinations of (i) and (ii).

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

9. A heterodimer comprising a first engineered cleavage half-domain of claim 1 and a second engineered cleavage half-domain.

10. The polypeptide of claim 1, further comprising a zinc finger DNA-binding domain.

11. A polynucleotide encoding the polypeptide of claim 1.

12. A polynucleotide encoding the polypeptide of claim 11.

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

14. An isolated cell comprising the polynucleotide of claim 15.

15. An isolated cell comprising the polynucleotide of claim 16.

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

(a) selecting a first nucleotide sequence in the region of interest;

(b) engineering a first zinc finger binding domain to bind to the first sequence;

(c) expressing a first fusion protein in a cell, the first fusion protein comprising the engineered zinc finger binding domain and an engineered cleavage half-domain of claim 1;

(d) expressing a second fusion protein in the cell, the second fusion protein comprising a second zinc finger binding domain engineered to bind to the second nucleotide sequence and a second engineered cleavage half-domain;

wherein the first fusion protein binds to the first nucleotide sequence, the second fusion protein binds to a second nucleotide sequence located between 2 and 50 nucleotides from the first nucleotide sequence, the first and second engineered cleavage domains form a heterodimer that cleaves the cellular chromatin in the region of interest.

17. The method of claim 16, wherein cleavage occurs between the first and second nucleotide sequences.

18. The method of claim 16, further comprising contacting the cell with a polynucleotide comprising a third nucleotide sequence, wherein the third nucleotide sequence is homologous but non-identical with the first nucleotide sequence;

wherein cleavage of the cellular chromatin in the region of interest facilitates homologous recombination between the first nucleotide sequence and the third nucleotide sequence, resulting in alteration of the first nucleotide sequence.

19. 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 zinc finger nucleases and further wherein each zinc finger nuclease comprises an engineered FokI cleavage domain according to claim 1.