Artificial nucleases comprising 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. An artificial nuclease comprising:
(i) a DNA-binding domain that binds to a target site in the genome of the cell; and
(ii) a polypeptide comprising an engineered FokI cleavage half-domain, wherein the engineered cleavage half-domain comprises substitution mutations 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. A method of making a genomic modification in a cell, the method comprising administering at least one artificial nuclease according to claim 1 to a cell, wherein the artificial nuclease genetically modifies a gene locus in the cell.
3. The method of claim 2 , wherein the gene locus is inactivated.
4. The method of claim 2 , wherein the genomic modification comprises an insertion.
5. The method of claim 4 , wherein a transgene is inserted into the gene locus.
6. The method of claim 2 , wherein the genomic modification comprises a deletion.
7. The method of claim 6 , wherein the genomic modification further comprises an insertion.
8. The method of claim 7 , wherein a transgene is inserted into the gene locus.
9. The method of claim 2 , wherein the cell is a T-cell, a hematopoietic stem cell or a liver cell.
10. The artificial nuclease 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.
11. The artificial nuclease 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.
12. The artificial nuclease 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.
13. The artificial nuclease 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.
14. The nuclease of claim 13 , further wherein the wild-type Ile (I) residue at position 499 is replaced with an Ala (A).
15. The artificial nuclease 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.
16. The artificial nuclease 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.
17. The artificial nuclease 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.
18. The artificial nuclease 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.