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-targeting systems comprising a novel DNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. Methods for making and using and uses of such systems, methods, and compositions and products from such methods and uses are also disclosed and claimed.
1. A modified Cpf1 effector protein comprising one or more mutations in the RuvC domain, wherein the modified Cpf1 effector protein has reduced catalytic activity.
2. The modified Cpf1 effector protein of claim 1 , wherein the modified Cpf1 effector protein comprises a mutation corresponding to amino acid position D917, E1006, or D1255 of Franscisella novicida U112 Cpf1.
3. The modified Cpf1 effector protein of claim 1 , wherein the modified Cpf1 effector protein is catalytically inactive.
4. The modified Cpf1 effector protein of claim 3 , wherein the modified Cpf1 effector protein is a modified Franscisella novicida Cpf1.
5. The modified Cpf1 effector protein of claim 4 , wherein the modified Cpf1 effector protein is a modified Franscisella novicida U112 Cpf1 comprising a mutation at D917 or E1006.
6. The modified Cpf1 effector protein of claim 5 , wherein the mutation is D917A or E1006A.
7. The modified Cpf1 effector protein of claim 3 , wherein the modified Cpf1 effector protein is a modified Acidaminococcus sp. Cpf1.
8. The modified Cpf1 effector protein of claim 7 , wherein the modified Cpf1 effector protein is a modified Acidaminococcus sp. BV3L6 Cpf1 comprising a mutation at D908, E993, or D1263.
9. The modified Cpf1 effector protein of claim 8 , wherein the mutation is D908A, E993A, or D1263A.
10. The modified Cpf1 effector protein of claim 3 , wherein the modified Cpf1 effector protein is a modified Lachnospiraceae bacterium Cpf1.
11. The modified Cpf1 effector protein of claim 10 , wherein the modified Cpf1 effector protein is a modified Lachnospiraceae bacterium ND2006 Cpf1 comprising a mutation at D832, E925, D947, or D1180.
12. The modified Cpf1 effector protein of claim 11 , wherein the mutation is D832A, E925A, D947A, or D1180A.
13. The modified Cpf1 effector protein of claim 3 , wherein the modified Cpf1 effector protein comprises one or more heterologous functional domains.
14. The modified Cpf1 effector protein of claim 13 , comprising one or more transcriptional activator domains.
15. The modified Cpf1 effector protein of claim 14 , wherein the transcriptional activator domain comprises VP64.
16. The modified Cpf1 effector protein of claim 13 , comprising one or more transcriptional repressor domains.
17. The modified Cpf1 effector protein of claim 16 , wherein the transcriptional repressor domain comprises KRAB or SID.
18. The modified Cpf1 effector protein of claim 13 , comprising one or more one or more nuclease domains.
19. The modified Cpf1 effector protein of claim 18 , wherein the nuclease domain comprises Fok1.
20. The modified Cpf1 effector protein of claim 13 , wherein the one or more heterologous functional domains have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity.
21. A composition comprising a CRISPR-Cpf1 complex, wherein the CRISPR-Cpf1 complex comprises the modified Cpf1 effector protein of claim 3 in complex with a guide polynucleotide comprising a guide sequence linked to a direct repeat sequence.
22. A method for binding a target sequence on a double-stranded DNA molecule, comprising exposing the double-stranded DNA molecule to the composition of claim 21 , wherein the guide polynucleotide directs sequence-specific binding of the CRISPR-Cpf1 complex to a target sequence on a target strand of the double-stranded DNA molecule.
23. A nucleic acid molecule comprising a nucleotide sequence encoding the modified Cpf1 effector protein of claim 3 .
24. A vector comprising a regulatory element operably linked to a nucleotide sequence encoding the modified Cpf1 effector protein of claim 3 .
25. An isolated cell comprising the modified Cpf1 effector protein of claim 3 .
26. The isolated cell of claim 25 , wherein the cell is a mammalian cell or a plant cell.
27. The modified Cpf1 effector protein of claim 1 , wherein the modified Cpf1 effector protein is a modified Cpf1 effector protein of Francisella tularensis 1 , Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1 , Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10 , Parcubacteria bacterium GW2011_GWC2_44_17 , Smithella sp. SCADC, Acidaminococcus sp. BV3L6 , Lachnospiraceae bacterium MA2020 , Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237 , Leptospira inadai, Lachnospiraceae bacterium ND2006 , Porphyromonas crevioricanis 3 , Prevotella disiens , or Porphyromonas macacae.