CRISPR enzymes and systems
The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.
1. A composition comprising:
a non-naturally occurring or engineered guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, wherein the gRNA is capable of forming a complex with a Cpf1 effector protein, wherein the gRNA comprises a loop comprising an aptamer sequence that is capable of binding to an adaptor protein; and
a mutated Cpf1 effector protein, wherein the mutated Cpf1 effector protein comprises at least one mutation, such that the mutated Cpf1 effector protein has no more than 5% of the nuclease activity of a corresponding wild-type Cpf1 effector protein not having the at least one mutation, and wherein the mutated Cpf1 effector protein comprises one or more nuclear localization sequences.
2. The composition of claim 1 , wherein the composition further comprises a fusion protein comprising an adaptor protein fused to one or more heterologous functional domains.
3. The composition of claim 2 , wherein the mutated Cpf1 effector protein has a diminished nuclease activity of at least 97%, or 100% as compared with the corresponding wild-type Cpf1 effector protein not having the at least one mutation.
4. The composition of claim 2 , wherein the mutated Cpf1 effector protein comprises at least one mutation selected from D908A, E993A, or D1263A according to AsCpf1 protein or a corresponding mutation in an ortholog.
5. The composition of claim 2 , wherein the mutated Cpf1 effector protein comprises at least one mutation selected from D832A, E925A, D947A or D1180A according to LbCpf1 protein or a corresponding mutation in an ortholog.
6. The composition of claim 5 , wherein the mutated Cpf1 effector protein comprises two or more mutations.
7. The composition of claim 2 , wherein the mutated Cpf1 effector protein is fused to one or more heterologous functional domains.
8. The composition of claim 2 , wherein the fusion protein comprises a linker between the adaptor protein and the heterologous functional domain, the linker optionally comprising a GlySer linker.
9. The composition of claim 2 , wherein the one or more heterologous functional domains fused to the adaptor protein comprise a transcriptional activation domain.
10. The composition of claim 7 , wherein the one or more heterologous functional domains fused to the mutated Cpf1 effector protein comprise a transcriptional activation domain.
11. The composition of claim 9 , wherein the transcriptional activation domain is VP64, p65, MyoD1, HSF1, RTA or SET7/9.
12. The composition of claim 10 , wherein the transcriptional activation domain is VP64, p65, MyoD1, HSF1, RTA or SET7/9.
13. The composition of claim 2 , wherein the one or more heterologous functional domains fused to the adaptor protein comprise a transcriptional repressor domain.
14. The composition of claim 7 , wherein the one or more heterologous functional domains fused to the mutated Cpf1 effector protein comprise a transcriptional repressor domain.
15. The composition of claim 13 , wherein the transcriptional repressor domain is a KRAB domain.
16. The composition of claim 13 , wherein the transcriptional repressor domain is a NuE domain, NcoR domain, SID domain or a SID4X domain.
17. The composition of claim 2 , wherein at least one of the one or more heterologous functional domains fused to the adaptor protein has one or more activities comprising methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, DNA integration activity RNA cleavage activity, DNA cleavage activity or nucleic acid binding activity.
18. The composition of claim 7 , wherein at least one of the one or more heterologous functional domains fused to the mutated Cpf1 effector protein has one or more activities comprising methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, DNA integration activity RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, or molecular switch activity or chemical inducibility or light inducibility.
19. The composition of claim 2 , wherein the adaptor protein is a bacteriophage coat protein selected from the group consisting of MS2, PP7, Qβ, GA, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s, and PRR1.
20. The composition of claim 2 , wherein the cell is a eukaryotic cell.
21. The composition of claim 20 , wherein the eukaryotic cell is a mammalian cell.
22. The composition of claim 21 , wherein the mammalian cell is a human cell.
23. The composition of claim 20 , wherein the eukaryotic cell is a plant cell.
24. A non-human transgenic cell or cell line which has been transformed with the composition according to claim 2 .
25. A non-human transgenic mammalian model, wherein the model has been transformed with the composition according to claim 2 .
26. An isolated host cell or cell line comprising the composition of claim 2 .
27. The isolated host cell or cell line according to claim 26 , wherein the cell is a eukaryotic cell.
28. The isolated host cell or cell line according to claim 26 , wherein the cell is an animal cell.
29. The isolated host cell or cell line according to claim 28 , wherein the cell is a human cell.
30. The isolated host cell or cell line according to claim 26 , comprising a stem cell or stem cell line.
31. The isolated host cell or cell line according to claim 26 , wherein the cell is a plant cell.
32. A vector system comprising one or more vector(s) comprising nucleic acid molecule(s) encoding: a guide RNA (gRNA) and a mutated Cpf1 effector protein,
wherein the mutated Cpf1 effector protein comprises at least one mutation, such that the mutated Cpf1 effector protein has no more than 5% of the nuclease activity of a corresponding wild-type Cpf1 effector protein not having the at least one mutation, and wherein the mutated Cpf1 effector protein comprises at least one or more nuclear localization sequences, and
wherein the gRNA is a non-naturally occurring or engineered gRNA comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, wherein the gRNA is capable of forming a complex with a Cpf1 effector protein, wherein the gRNA comprises a loop comprising an aptamer sequence that is capable of binding to an adaptor protein.
33. The vector system of claim 32 , nucleic acid molecule(s) further encoding a fusion protein comprising an adaptor protein fused to one or more heterologous functional domains.