Type VI CRISPR orthologs and systems
View Patent ↗The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.
1 . A non-naturally occurring or engineered composition for modifying a target RNA sequence, said composition comprising:
(a) a Cas13b effector protein optimized for activity in a mammalian cell,
(b) a guide molecule capable of forming a complex with the Cas13b and directing sequence-specific binding of the complex to a target sequence of a target RNA, wherein the target RNA sequence comprises one or more genes associated with disease, and
(c) one or more heterologous functional domains fused to the Cas13b effector protein, wherein the one or more heterologous functional domains modifies the target RNA sequence,
wherein the Cas13b effector protein is at least 99% homologous to the Cas13b effector protein selected from the group consisting of SEQ ID NO: 146-156, 158-163, and 165-166.
2 . The composition of claim 1 , wherein the Cas13b effector protein is catalytically inactive.
3 . The composition of claim 1 , wherein the Cas13b effector protein comprises one to nine mutations to the two HEPN domains.
4 . The composition of claim 1 , wherein the Cas13b effector protein comprises a mutation in one or more of positions corresponding to R116A, H121A, R1177A, and H1182A of Cas13b effector protein originating from Bergeyella zoohelcum ATCC 43767 as set forth in SEQ ID NO: 577.
5 . The composition of claim 1 , wherein the Cas13b effector protein corresponds to amino acids 1-984 of Prevotella sp. P5-125 Cas13b as set forth in SEQ ID NO: 160.
6 . The composition of claim 1 , wherein the guide molecule hybridizes to a target RNA sequence comprising an adenine to form an RNA duplex, wherein the guide molecule comprises a non-pairing cytosine at a position corresponding to said adenine, resulting in an A-C mismatch in the RNA duplex formed.
7 . The composition of claim 1 , wherein the guide molecule comprises more than one mismatch corresponding to different adenosine sites in the target RNA sequence or wherein two guide molecules are used, each comprising a mismatch corresponding to a different adenosine site in the target RNA sequence.
8 . The composition of claim 1 , wherein the one or more heterologous functional domains modifies the target RNA sequence by converting adenosine to inosine.
9 . The composition of claim 8 , wherein the one or more heterologous functional domains comprises an adenosine deaminase.
10 . The composition of claim 9 , wherein the adenosine deaminase is fused to a N- or C-terminus of the Cas13b effector protein.
11 . The composition of claim 10 , wherein the adenosine deaminase is fused by a linker.
12 . The composition of claim 11 , wherein the linker is (GGGGS) 3-11 , GSG 5 , or LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 145), or wherein the linker is an XTEN linker.
13 . The composition of claim 9 , wherein the adenosine deaminase is linked to an adaptor protein and the guide molecule.
14 . The composition of claim 10 , wherein the Cas13b effector protein comprises an aptamer sequence capable of binding to an adaptor protein, wherein the adaptor protein is selected from MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, and PRR1.
15 . The composition of claim 9 , wherein the adenosine deaminase is an RNA-specific adenosine deaminase or catalytic domain thereof.
16 . The composition of claim 15 , wherein the RNA-specific adenosine deaminase is ADAR.
17 . The composition of claim 16 , wherein the ADAR is human ADAR (huADAR) and/or ADAR1 or ADAR2, or a catalytic domain thereof.
18 . The composition of claim 17 , wherein the ADAR is huADAR or a catalytic domain thereof.
19 . The composition of claim 16 , wherein the ADAR is a mutated hADAR2d comprising mutation E488Q or a mutated hADAR1d comprising mutation E1008Q.
20 . The composition of claim 1 , wherein the one or more genes associated with disease comprise one or more of PCSK9, HBB, AVPR2, FANCC, IL2RG, F8, LDLR, CBS, ALDOB, DMD, SMAD4, BRCA1, BRCA2, GRIN2A, SCN9A, TARDBP, CFTR, UBE3A, SMPD1, USH2A, MEN1, C8orf37, MLH1, TSC2, NF1, MSH6, SMN1, SH3TC2, DNAH5, MECP2, ADGRV1, AHI1, PRKN, COL3A1, MYBPC3, APC, and BMPR2.
21 . The composition of claim 20 , wherein the one or more genes associated with disease is PCSK9.
22 . The composition of claim 20 , wherein the one or more genes associated with disease is HBB.
23 . The composition of claim 1 , wherein the Cas13b effector protein comprises one or more heterologous nuclear export signals (NES) or nuclear localization signals (NLS).
24 . The composition of claim 23 , wherein the NES is an HIV Rev NES or MAPK NES.
25 . The composition of claim 23 , wherein the heterologous NES or NLS is fused at the C-terminal of the Cas13b effector protein.
26 . A mammalian cell comprising the composition of claim 1 .
27 . A cell line comprising the mammalian cell of claim 26 , or progeny thereof.