	US 11,725,218 B2
	Artificial nucleases comprising engineered cleavage half-domains
Yannick Doyon, Brisbane, CA (US); and Jeffrey C. Miller, Brisbane, CA (US)
Assigned to Sangamo Therapeutics, Inc., Brisbane, CA (US)
Filed by Sangamo Therapeutics, Inc., Brisbane, CA (US)
Filed on Oct. 4, 2021, as Appl. No. 17/493,655.
Application 17/493,655 is a continuation of application No. 16/838,804, filed on Apr. 2, 2020, granted, now 11,136,599.
Application 16/838,804 is a continuation of application No. 16/055,525, filed on Aug. 6, 2018, granted, now 10,640,790, issued on May 5, 2020.
Application 16/055,525 is a continuation of application No. 15/660,072, filed on Jul. 26, 2017, granted, now 10,066,242, issued on Sep. 4, 2018.
Application 15/660,072 is a continuation of application No. 15/160,571, filed on May 20, 2016, granted, now 9,765,361, issued on Sep. 19, 2017.
Application 15/160,571 is a continuation of application No. 14/627,812, filed on Feb. 20, 2015, granted, now 9,376,689, issued on Jun. 28, 2016.
Application 14/627,812 is a continuation of application No. 12/931,660, filed on Feb. 7, 2011, granted, now 8,962,281, issued on Feb. 24, 2015.
Claims priority of provisional application 61/337,769, filed on Feb. 8, 2010.
Claims priority of provisional application 61/403,916, filed on Sep. 23, 2010.
Prior Publication US 2022/0145307 A1, May 12, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/90 (2006.01); C12N 9/22 (2006.01); C12N 9/10 (2006.01); C12N 15/52 (2006.01); C12N 15/85 (2006.01); C07K 14/47 (2006.01); C07K 7/06 (2006.01)

CPC C12N 15/907 (2013.01) [C07K 7/06 (2013.01); C07K 14/47 (2013.01); C12N 9/10 (2013.01); C12N 9/22 (2013.01); C12N 15/52 (2013.01); C12N 15/85 (2013.01); C07K 2319/50 (2013.01); C07K 2319/80 (2013.01); C07K 2319/81 (2013.01); C12Y 301/21004 (2013.01)]

18 Claims

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.