US 11,873,504 B2
RNA-guided nucleic acid modifying enzymes and methods of use thereof
Jennifer A Doudna, Berkeley, CA (US); Jillian F. Banfield, Berkeley, CA (US); David Burstein, Berkeley, CA (US); Lucas Benjamin Harrington, Berkeley, CA (US); and Steven C. Strutt, Berkeley, CA (US)
Assigned to The Regents of the University of California, Oakland, CA (US)
Appl. No. 16/335,516
Filed by The Regents of the University of California, Oakland, CA (US)
PCT Filed Sep. 28, 2017, PCT No. PCT/US2017/054081
§ 371(c)(1), (2) Date Mar. 21, 2019,
PCT Pub. No. WO2018/064371, PCT Pub. Date Apr. 5, 2018.
Claims priority of provisional application 62/402,846, filed on Sep. 30, 2016.
Prior Publication US 2020/0017879 A1, Jan. 16, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/85 (2006.01); C12N 9/22 (2006.01); C12N 15/113 (2010.01); C12N 15/10 (2006.01); C12N 15/86 (2006.01); C12N 15/90 (2006.01); C07K 19/00 (2006.01); C12N 9/10 (2006.01); C12N 15/52 (2006.01); C12N 15/62 (2006.01); C12N 15/74 (2006.01); C12N 15/88 (2006.01)
CPC C12N 15/8509 (2013.01) [C07K 19/00 (2013.01); C12N 9/1007 (2013.01); C12N 9/1025 (2013.01); C12N 9/22 (2013.01); C12N 15/102 (2013.01); C12N 15/113 (2013.01); C12N 15/52 (2013.01); C12N 15/62 (2013.01); C12N 15/74 (2013.01); C12N 15/85 (2013.01); C12N 15/86 (2013.01); C12N 15/88 (2013.01); C12N 15/902 (2013.01); C07K 2319/02 (2013.01); C07K 2319/03 (2013.01); C07K 2319/06 (2013.01); C07K 2319/09 (2013.01); C07K 2319/20 (2013.01); C12N 2310/20 (2017.05); C12N 2740/13043 (2013.01); C12N 2740/16043 (2013.01); C12N 2750/14143 (2013.01)] 31 Claims
 
1. One or more nucleic acid molecules encoding one or more of:
(a) a CasX guide RNA comprising an activator RNA and a targeter RNA, wherein the targeter RNA is heterologous to the activator RNA,
wherein one nucleic molecule comprises a first nucleotide sequence encoding the activator RNA and a second nucleotide sequence encoding the targeter RNA, wherein the first nucleotide sequence is heterologous to the second nucleotide sequence, or
wherein a first nucleic molecule comprises a nucleotide sequence encoding the activator RNA and a second nucleic molecule comprises a nucleotide sequence encoding the targeter RNA, wherein the first nucleic molecule is heterologous to the second nucleic molecule,
and
wherein the targeter RNA comprises (i) a guide sequence that hybridizes to a target nucleic acid, and (ii) a duplex-forming segment and the activator RNA hybridizes with the duplex-forming segment to form a double stranded RNA (dsRNA) duplex that binds a CasX polypeptide and guides the bound CasX polypeptide to the target nucleic acid;
(b) a CasX polypeptide, wherein the one nucleic acid molecule comprises a promoter operably linked to and heterologous to the sequence encoding the CasX polypeptide; and
(c) a CasX fusion polypeptide comprising a CasX polypeptide fused to a heterologous polypeptide, wherein the one nucleic acid molecule comprises a nucleotide sequence encoding the CasX fusion polypeptide.