US 12,365,887 B2
CRISPR-Cas effector polypeptides and methods of use thereof
Jennifer A. Doudna, Berkeley, CA (US); Basem Al-Shayeb, Berkeley, CA (US); Jillian F. Banfield, Berkeley, CA (US); and Patrick Pausch, Berkeley, CA (US)
Assigned to The Regents of the University of California, Oakland, CA (US)
Filed by The Regents of the University of California, Oakland, CA (US)
Filed on May 5, 2021, as Appl. No. 17/308,568.
Application 17/308,568 is a continuation of application No. PCT/US2020/021213, filed on Mar. 5, 2020.
Claims priority of provisional application 62/948,470, filed on Dec. 16, 2019.
Claims priority of provisional application 62/907,422, filed on Sep. 27, 2019.
Claims priority of provisional application 62/855,739, filed on May 31, 2019.
Claims priority of provisional application 62/815,173, filed on Mar. 7, 2019.
Prior Publication US 2021/0324358 A1, Oct. 21, 2021
Int. Cl. C12N 9/22 (2006.01); A61K 31/7088 (2006.01); A61K 38/00 (2006.01); A61K 38/46 (2006.01); A61K 48/00 (2006.01); C12N 15/10 (2006.01); C12N 15/11 (2006.01); C12N 15/90 (2006.01); C12Q 1/6818 (2018.01)
CPC C12N 9/22 (2013.01) [A61K 31/7088 (2013.01); A61K 38/465 (2013.01); C12N 15/102 (2013.01); C12N 15/11 (2013.01); C12N 15/111 (2013.01); C12N 15/907 (2013.01); C12Q 1/6818 (2013.01); A61K 38/00 (2013.01); A61K 48/00 (2013.01); C07K 2319/02 (2013.01); C07K 2319/09 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] 23 Claims
 
1. A method of cleaving double stranded DNA (dsDNA) in a cell, the method comprising contacting the cell with:
a) a nucleic acid encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 109-126; and
b) a guide nucleic acid, wherein the guide nucleic acid comprises:
i) a repeat sequence; and
ii) a spacer sequence, wherein the spacer sequence hybridizes to a target sequence on a target strand of the dsDNA, wherein:
the target sequence is adjacent to a protospacer adjacent motif (PAM) of 5′-NTTN-3′ and the PAM is located 5′ of the target sequence on the non-target strand, and
the polypeptide and the guide nucleic acid form a ribonucleoprotein (RNP) complex that binds the target sequence and cleaves the dsDNA.
 
15. A composition for cleaving double stranded DNA (dsDNA) in a cell, the composition comprising:
a) a nucleic acid encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 109-126; and
b) a guide nucleic acid, wherein the guide nucleic acid comprises:
i) a repeat sequence; and
ii) a spacer sequence, wherein the spacer sequence hybridizes to a target sequence on a target strand of the dsDNA, wherein:
the target sequence is adjacent to a protospacer adjacent motif (PAM) of 5′-NTTN-3′ and the PAM is located 5′ of the target sequence on the non-target strand, and
the polypeptide and the guide nucleic acid form a ribonucleoprotein (RNP) complex that binds the target sequence and cleaves the dsDNA.