	US 12,215,326 B2
	RNA-targeting system
Feng Zhang, Cambridge, MA (US); Patrick Hsu, Cambridge, MA (US); Jonathan S. Gootenberg, Cambridge, MA (US); and Aaron Smargon, Cambridge, MA (US)
Assigned to The Broad Institute, Inc., Cambridge, MA (US); Massachusetts Institute of Technology, Cambridge, MA (US); and President and Fellows of Harvard College, Cambridge, MA (US)
Filed by The Broad Institute, Inc., Cambridge, MA (US); Massachusetts Institute of Technology, Cambridge, MA (US); and President and Fellows of Harvard College, Cambridge, MA (US)
Filed on Jun. 23, 2017, as Appl. No. 15/632,067.
Application 15/632,067 is a continuation in part of application No. PCT/US2015/067151, filed on Dec. 21, 2015.
Claims priority of provisional application 62/096,324, filed on Dec. 23, 2014.
Claims priority of provisional application 62/098,059, filed on Dec. 30, 2014.
Claims priority of provisional application 62/181,641, filed on Jun. 18, 2015.
Claims priority of provisional application 62/181,667, filed on Jun. 18, 2015.
Prior Publication US 2017/0306335 A1, Oct. 26, 2017
Int. Cl. C12N 15/63 (2006.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12N 15/115 (2010.01); C12N 15/82 (2006.01); C12N 15/86 (2006.01)

CPC C12N 15/63 (2013.01) [C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 15/111 (2013.01); C12N 15/115 (2013.01); C12N 15/86 (2013.01); C12Y 301/21004 (2013.01); C07K 2319/09 (2013.01); C12N 2310/10 (2013.01); C12N 2310/16 (2013.01); C12N 2310/20 (2017.05); C12N 2330/51 (2013.01); C12N 2800/22 (2013.01)]

36 Claims

1. A non-naturally occurring or engineered vector system comprising one or more vectors comprising:

a) a first regulatory element operably linked to a first polynucleotide sequence encoding an RNA-targeting guide RNA (Rt-gRNA), wherein said Rt-gRNA is designed to hybridize with a target RNA, wherein said Rt-gRNA comprises:

(i) a small CRISPR/Cas system associated RNA (scaRNA) sequence, and

(ii) a trans-activating CRISPR/Cas system RNA (tracrRNA) sequence, wherein said scaRNA and said tracrRNA are designed to at least partially hybridize to each other, and

b) a second regulatory element operably linked to a second polynucleotide sequence encoding

(i) a Francisella novicida Cas protein (FnCas) or ortholog, homolog, or fragment thereof comprising a Rec1 domain, Rec2 domain, HNH domain, and PAM Interacting (PI) domain, and

(ii) one or more heterologous functional domains positioned at one or more sites in FnCas that are analogous to amino acid positions 534-676 corresponding to a wild type Streptococcus pyogenes Cas9 (SpCas9) Rec1 domain

wherein the Rt-gRNA is capable of forming an RNA-targeting complex with the FnCas and of directing sequence-specific binding of the RNA-targeting complex to the target RNA,

wherein each of the one or more heterologous functional domains comprise one or more of the following activities: methylase activity, demethylase activity, translation activation activity, translation repression activity, histone modification activity, RNA cleavage activity, or DNA cleavage activity, and

wherein components (a) and (b) are located on the same or different vectors of the system and wherein the Rt-gRNA and said FnCas protein do not naturally occur together.