Cpf1 complexes with reduced indel activity
The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA-targeting systems comprising a novel DNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.
1. An engineered guide RNA (gRNA) capable of forming a CRISPR-Cpf1 complex with a Cpf1 effector protein and directing the CRISPR-Cpf1 complex to a target sequence;
wherein the gRNA comprises a dead guide sequence capable of reducing indel activity of the CRISPR-Cpf1 complex;
wherein said guide sequence comprises one or more mismatches to the target sequence in a seed region at nucleotide position 14-18 of the guide sequence.
2. The gRNA of claim 1 , wherein indel activity is reduced by at least 90%, or at least 95%, or at least 97%, or at least 99%.
3. The gRNA of claim 1 , wherein indel activity is detected by a SURVEYOR assay.
4. The gRNA of claim 1 , wherein indel activity is detected by a BLISS assay.
5. The gRNA of claim 1 , wherein said guide sequence comprises a matching section and a mismatching section with the matching section being 12-15 contiguous nucleotides in length and complementary to the target sequence.
6. The gRNA of claim 1 , wherein the guide sequence comprises 10-16 contiguous nucleotides complementary to the target sequence immediately adjacent to a PAM sequence.
7. The gRNA of claim 1 , wherein the guide sequence comprises 2 or more mismatches to the target sequence.
8. The gRNA of claim 1 , wherein the guide sequence comprises 3 or more mismatches to the target sequence.
9. The gRNA of claim 1 , wherein the guide sequence comprises 1, 2, or 3 mismatches to the target sequence.
10. A composition comprising the gRNA of claim 1 , and further comprising a Cpf1 effector protein capable of forming a CRISPR-Cpf1 complex with the gRNA.
11. The composition of claim 10 , wherein the Cpf1 is FnCpf1, AsCpf1, or LbCpf1.
12. The composition of claim 10 , wherein the Cpf1 effector protein comprises at least one mutation.
13. The composition of claim 10 , wherein the Cpf1 effector protein comprises a nickase.
14. The composition of claim 10 , wherein the Cpf1 effector protein comprises one or more nuclear localization signals.
15. The composition of claim 10 , further comprising one or more adaptor proteins, wherein at least one loop of the gRNA comprises an aptamer sequence, wherein each adaptor protein is associated with one or more functional domains, and wherein the adaptor protein is capable of binding to the aptamer sequence.
16. The composition of claim 15 , wherein the adaptor protein comprises 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, or PRR1.
17. The composition of claim 10 , wherein the Cpf1 is associated with one or more functional domains.
18. The composition of claim 15 , wherein the one or more functional domains comprises an activator domain, a repressor domain, a recombinase, a transposase, a histone remodeler, a DNA methyltransferase, a cryptochrome, a deaminase, a light inducible/controllable domain, or a chemically inducible/controllable domain.
19. The composition of claim 15 , wherein the one or more functional domains is a transcriptional activation domain and comprises VP64, p65, MyoD1, HSF1, RTA, or SETT/9.
20. The composition of claim 15 , wherein the one or more functional domains is a transcriptional repressor domain and comprises a KRAB domain, NuE domain, NcoR domain, SID domain, or a SID4X domain.
21. The composition of claim 10 , further comprising a second gRNA capable of forming a second CRISPR-Cpf1 complex with a Cpf1 effector protein and directing the second CRISPR-Cpf1 complex to a second target sequence, wherein the second gRNA comprises a guide sequence capable of hybridizing to the second target sequence while permitting detectable indel activity by the second CRISPR-Cpf1 complex.
22. The composition of claim 10 , comprising a plurality of guide gRNAs each having a dead guide sequence.
23. A cell comprising the composition of claim 10 , wherein the cell is a eukaryotic cell.
24. A particle comprising the composition of claim 10 .
25. A method for modifying a genomic locus, which comprises administering the composition of claim 10 to a eukaryotic cell comprising the genomic locus.
26. The method according to claim 25 , wherein the genomic locus is modified by transcriptional gene activation, transcriptional gene inhibition, or cleavage in the genomic locus.
27. A vector system comprising a nucleic acid molecule encoding the gRNA of claim 1 .
28. The vector system of claim 27 , further comprising a nucleic acid molecule encoding the Cpf1 effector protein.