Engineered cleavage half-domains
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.
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 .