US 11,674,159 B2
Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
Jennifer A. Doudna, Berkeley, CA (US); Martin Jinek, Berkeley, CA (US); Krzysztof Chylinski, Vienna (AT); and Emmanuelle Charpentier, Berlin (DE)
Assigned to The Regents of the University of California, Oakland, CA (US); University of Vienna, Vienna (AT); and Emmanuelle Charpentier, Berlin (DE)
Filed by The Regents of the University of California, Oakland, CA (US); University of Vienna, Vienna (AT); and Emmanuelle Charpentier, Berlin (DE)
Filed on Apr. 1, 2021, as Appl. No. 17/220,692.
Application 17/220,692 is a continuation of application No. 16/385,360, filed on Apr. 16, 2019.
Application 16/385,360 is a continuation of application No. 13/842,859, filed on Mar. 15, 2013, granted, now 10,266,850, issued on Apr. 23, 2019.
Claims priority of provisional application 61/765,576, filed on Feb. 15, 2013.
Claims priority of provisional application 61/757,640, filed on Jan. 28, 2013.
Claims priority of provisional application 61/716,256, filed on Oct. 19, 2012.
Claims priority of provisional application 61/652,086, filed on May 25, 2012.
Prior Publication US 2021/0222205 A1, Jul. 22, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/90 (2006.01); C12N 15/11 (2006.01); C12N 15/63 (2006.01); C12N 9/22 (2006.01); C12N 15/113 (2010.01); C12N 15/10 (2006.01); A61K 38/46 (2006.01); C12Q 1/686 (2018.01); H01L 21/205 (2006.01); H01L 33/00 (2010.01); H01L 21/02 (2006.01); C12N 15/70 (2006.01); C12N 15/74 (2006.01); A01H 6/46 (2018.01); A01K 67/027 (2006.01); A61K 48/00 (2006.01)
CPC C12N 15/907 (2013.01) [A01H 6/4684 (2018.05); A01K 67/027 (2013.01); A61K 38/465 (2013.01); C12N 9/22 (2013.01); C12N 15/102 (2013.01); C12N 15/111 (2013.01); C12N 15/113 (2013.01); C12N 15/63 (2013.01); C12N 15/70 (2013.01); C12N 15/746 (2013.01); C12N 15/90 (2013.01); C12N 15/902 (2013.01); C12Q 1/686 (2013.01); H01L 21/02312 (2013.01); H01L 21/205 (2013.01); H01L 33/0075 (2013.01); A61K 48/00 (2013.01); C12N 2310/11 (2013.01); C12N 2310/13 (2013.01); C12N 2310/14 (2013.01); C12N 2310/20 (2017.05); C12N 2310/31 (2013.01); C12N 2310/32 (2013.01); C12N 2310/33 (2013.01); C12N 2310/3519 (2013.01); C12N 2310/531 (2013.01); C12N 2800/80 (2013.01); C12Y 301/04 (2013.01)] 165 Claims
 
1. A method of producing a modified prokaryotic cell, the method comprising: introducing into a target prokaryotic cell comprising a target DNA:
(a) a chimeric Cas9 protein, or a nucleic acid encoding the chimeric Cas9 protein, wherein the chimeric Cas9 protein comprises a Cas9 polypeptide fused to a heterologous polypeptide, wherein the Cas9 polypeptide comprises one or more mutations in a RuvC domain and/or an HNH domain; and
(b) a DNA-targeting RNA that comprises:
(i) a targeter-RNA that is capable of hybridizing with a target sequence of the target DNA, and
(ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded duplex,
wherein (i) and (ii) are covalently linked,
wherein the DNA-targeting RNA targets the chimeric Cas9 protein to the target sequence of the target DNA, and wherein said introducing produces a modified prokaryotic cell.