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. An engineered CRISPR-Cas guide polynucleotide comprising, from 5′ to 3′, a direct repeat sequence linked to a heterologous guide sequence, wherein the guide sequence is heterologous to the direct repeat sequence;
wherein the guide sequence is engineered to hybridize with a target sequence in a eukaryotic cell, wherein the direct repeat sequence comprises a stem-loop region; and
wherein the guide polynucleotide is capable of forming a CRISPR-Cas complex with a Type V-A Cas protein and directing sequence-specific binding of the CRISPR-Cas complex to the target sequence in the eukaryotic cell.
2. The engineered CRISPR-Cas guide polynucleotide of claim 1 , wherein the direct repeat sequence comprises, from 5′ to 3′, a stem left region, a loop region, a stem right region, and an Uracil immediately 5′ of the guide sequence.
3. The engineered CRISPR-Cas guide polynucleotide of claim 2 , wherein the stem left region and the stem right region comprise the sequences, respectively, of UCUAC and GUAGA.
4. The engineered CRISPR-Cas guide polynucleotide of claim 2 , wherein the stem left region and the stem right region comprise the sequences, respectively, of CCUAC and GUAGG, UCCAC and GUGGA, or UCUGC and GCAGA.
5. The engineered CRISPR-Cas guide polynucleotide of claim 2 , wherein the loop region comprises the sequence of UGUU or UAUU.
6. The engineered CRISPR-Cas guide polynucleotide of claim 2 , wherein the loop region comprises the sequence of UCUU, UUUU, UGCU, UUCG, UCUUU, UGUUU, UAAGU, UUU or UU.
7. The engineered CRISPR-Cas guide polynucleotide of claim 2 , wherein the direct repeat sequence further comprises AAUU or AUU immediately 5′ of the stem left region.
8. The engineered CRISPR-Cas guide polynucleotide of claim 2 , wherein the direct repeat sequence further comprises GG at the 5′ end.
9. The engineered CRISPR-Cas guide polynucleotide of claim 2 , wherein the stem left region and the stem right region comprise the sequences, respectively, of UCUAC and GUAGA; wherein the loop region comprises the sequence of UGUU, UAUU, UCUU or UUUU; wherein the direct repeat sequence comprises AAUU or AUU immediately 5′ of the stem left region.
10. The engineered CRISPR-Cas guide polynucleotide of claim 1 , wherein the guide sequence is 17-24 nucleotides in length, and wherein the direct repeat sequence is 16-21 nucleotides in length.
11. The engineered CRISPR-Cas guide polynucleotide of claim 1 , wherein the direct repeat sequence is any of SEQ ID NOs:1434, 1437-1438 and 1440-1449, or is encoded by any of SEQ ID NOs:195, 198-199 and 201-210.
12. The engineered CRISPR-Cas guide polynucleotide of claim 1 , wherein the engineered CRISPR-Cas guide polynucleotide comprises at least one chemical modification.
13. The engineered CRISPR-Cas guide polynucleotide of claim 12 , wherein the chemical modification comprises pseudo-U, 5-methyl-C, a methylated nucleotide, or a nucleotide analog.
14. The engineered CRISPR-Cas guide polynucleotide of claim 12 , wherein the chemical modification comprises 2′-O-methyl, 2′-O-methyl 3′ phosphorothioate, or 2′-O-methyl 3′ thioPACE.
15. The engineered CRISPR-Cas guide polynucleotide of claim 12 , wherein the chemical modification comprises a phosphorothioate linkage or a methylene bridge between 2′ and 4′ carbons of a ribose ring.
16. The engineered CRISPR-Cas guide polynucleotide of claim 1 , wherein the target sequence is adjacent to a protospacer motif (PAM) in the genome of the eukaryotic cell, wherein the PAM comprises 5′-TTN or 5′-TTTV.
17. The engineered CRISPR-Cas guide polynucleotide of claim 1 , wherein the guide polynucleotide is capable of forming a CRISPR-Cas complex with a Type V-A Cas protein without any tracrRNA.
18. A composition comprising (a) a Type V-A Cas protein or a nucleic acid encoding the Type V-A Cas protein and (b) the engineered CRISPR-Cas guide polynucleotide of claim 1 or a nucleic acid encoding the engineered CRISPR-Cas guide polynucleotide.
19. The composition of claim 18 , wherein the composition comprises a ribonucleoprotein comprising the engineered CRISPR-Cas guide polynucleotide in complex with the Type V-A Cas protein.
20. The composition of claim 18 , wherein the composition comprises a lipid particle comprising the engineered CRISPR-Cas guide polynucleotide and an mRNA encoding the Type V-A Cas protein.
21. The composition of claim 18 , wherein the composition comprises one or more viral vectors comprising or encoding the engineered CRISPR-Cas guide polynucleotide and the Type V-A Cas protein.
22. The composition of claim 18 , wherein the Type V-A Cas protein does not comprise a HNH domain and comprises RuvC-I, RuvC-II, and RuvC-III domains.
23. The composition of claim 22 , wherein the Type V-A Cas protein comprises a VIGIDRG (SEQ ID NO:1596) or IIGIDRG (SEQ ID NO:1597) motif in the RuvC-I domain.
24. The composition of claim 22 , wherein the Type V-A Cas protein comprises a DANGAY (SEQ ID NO:1598) motif in the RuvC-III domain.
25. The composition of claim 18 , wherein the Type V-A Cas protein is fused to one or more heterologous nuclear localization signals.
26. The composition of claim 18 , wherein the Type V-A Cas protein comprises at least one mutation in a catalytic domain and has reduced catalytic activity.
27. The composition of claim 26 , wherein the Type V-A Cas protein is fused to one or more heterologous protein domains.
28. The composition of claim 27 , wherein the one or more heterologous protein domains are selected from the group consisting of transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA hydroxylmethylase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, transcriptional repressor domain, transcriptional activator domain, deaminase domain, transcription-regulatory protein domain, cellular uptake activity associated domain, nucleic acid binding domain, antibody presentation domain, histone modifying enzyme, recruiter of histone modifying enzyme, inhibitor of histone modifying enzyme, histone methyltransferase, histone demethylase, histone kinase, histone phosphatase, histone ribosylase, histone deribosylase, histone ubiquitinase, histone deubiquitinase, histone biotinase, and histone tail protease.
29. The composition of claim 18 , wherein the Type V-A Cas protein is obtained from Francisella, Prevotella, Acidaminococcus, Lachnospiraceae, Moraxella, Eubacterium, Smithella, Butyrivibrio, Peregrinibacteria, Parcubacteria, Candidatus, Leptospira , or Porphyromonas.
30. The composition of claim 18 , further comprising a donor template for targeted integration of a transgene by homology-directed repair.