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 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 method of modifying a target locus of interest, the method comprising: delivering to said target locus of interest a non-naturally occurring or engineered composition comprising a Type VI Cas polypeptide comprising two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains and one or more nucleic acid components, wherein the Cas polypeptide forms a complex with the one or more nucleic acid components, the one or more nucleic acid components directing binding of the complex to the target locus of interest thereby modifying the target locus of interest.
2. The method of claim 1 , wherein the target locus of interest is provided via a nucleic acid molecule within a cell.
3. The method of claim 2 , wherein the cell is a prokaryotic cell or a eukaryotic cell.
4. The method of claim 2 , wherein when in complex with the Cas polypeptide, the nucleic acid component(s) is a guide RNA capable of effecting sequence-specific binding of the complex to a target sequence of the target locus of interest.
5. The method of claim 4 , wherein the nucleic acid component(s) comprise a dual direct repeat sequence.
6. The method of claim 4 , wherein the guide RNA does not comprise a tracr sequence.
7. The method of claim 4 , wherein the target sequence is a disease associated RNA.
8. The method of claim 4 , wherein the target sequence is a disease-specific RNA.
9. The method of claim 1 , wherein the Cas polypeptide and nucleic acid component(s) are provided via one or more polynucleotide molecules encoding the polypeptide and/or the nucleic acid component(s), and wherein the one or more polynucleotide molecules are operably configured to express the polypeptide and/or the nucleic acid component(s).
10. The method of claim 9 , wherein the one or more polynucleotide molecules comprise one or more regulatory elements operably configured to express the polypeptides and/or the nucleic acid component(s), optionally wherein the one or more regulatory elements comprise a promoter(s) or inducible promotor(s).
11. The method of claim 9 , wherein the one or more polynucleotide molecules are comprised within one or more vectors.
12. The method of claim 11 , wherein the one or more vectors comprise one or more viral vectors.
13. The method of claim 12 , wherein the one or more viral vectors comprise one or more retroviral, lentiviral, adenoviral, adeno-associated or herpes simplex viral vectors.
14. The method of claim 9 , wherein the one or more polynucleotide molecules are comprised within one vector.
15. The method of claim 1 , wherein the non-naturally occurring or engineered composition is delivered via liposomes, nanoparticles, exosomes, microvesicles, a gene-gun or one or more viral vectors.
16. The method of claim 1 , wherein modifying is or comprises a nucleotide strand break.
17. The method of claim 1 , wherein the target locus of interest comprises RNA.
18. The method of claim 1 , wherein the target locus of interest comprises a target sequence that is at least 83% complementary to the one or more nucleic acid components.
19. The method of claim 1 , wherein the Cas polypeptide is a C2c2 polypeptide.
20. The method of claim 19 , wherein the C2c2 polypeptide is selected from a bacteria belonging to a genus selected from the group consisting of: Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconocetobacter, Neisseria, Roseburia, Porvibaculum, Staphylococcus, Nitratifactor, Camplyobacter, Leptotrichia, Rhodobacter , Lachnospiraceae, Carnobacterium , and Paludibacter.
21. The method of claim 20 , wherein the C2c2 polypeptide is an orthologue comprising one or more HEPN domains, the one or more HEPN domains comprise a RxxxxH catalytic motif, and the one or more HEPN domains comprise at least 95% sequence identity to one of SEQ ID NO: 512-547.
22. The method of claim 21 , wherein the C2c2 polypeptide is selected from the group consisting of SEQ ID NO. 573-587 and 591.
23. The method of claim 19 , wherein the C2c2 polypeptide is codon optimized for expression in a eukaryotic cell.
24. The method of claim 19 , wherein the C2c2 polypeptide is associated with one or more functional domains; and optionally the polypeptide contains one or more mutations optionally within an HEPN Domain, the one or more mutations comprising R597A, H602A, R1278A, and/or H1283A, whereby the complex can deliver an epigenetic modifier or a transcriptional or translational activation or repression signal to the target locus of interest.
25. The method of claim 19 , wherein the C2c2 polypeptide comprises at least one or more nuclear localization signals.
26. An in vitro method for modifying a RNA comprising performing the method of claim 1 .
27. The method of claim 26 , further comprising detecting binding of the complex to the RNA.