Type VI CRISPR orthologs 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 non-naturally occurring or engineered composition comprising i) a Cas13a CRISPR-Cas effector protein fused to one or more localization signals, or one or more polynucleotide molecules encoding a Cas13a CRISPR-Cas effector protein fused to one or more localization signals; and ii) one or more nucleic acid components, the one or more nucleic acid components comprising a heterologous guide sequence that is capable of hybridizing to a target RNA sequence, whereby the effector protein forms a complex with the one or more nucleic acid components, the one or more nucleic acid components directs the complex to the target RNA sequence in a mammalian target locus and the complex binds to the target RNA sequence;
wherein the Cas13a CRISPR-Cas effector protein is selected from Cas13a CRISPR-Cas proteins obtained from Eubacteriaceae bacterium CHKCI004 , Blautia sp. Marseille-P2398, RNA-binding protein S1 Chloroflexus aggregans, Demequina aurantiaca, Thalassospira sp. TSL5-1, SAMN04487830_13920 Pseudobutyrivibrio sp. OR37, SAMN02910398_00008 Butyrivibrio sp. YAB3001 , Leptotrichia sp. Marseille-P3007, Bacteroides ihuae , SAMN05216357_1045 Porphyromonadaceae bacterium KH3CP3RA, Listeria riparia , and Insolitispirillum peregrinum.
2. The composition of claim 1 , wherein the Cas 13a effector protein comprises at least two Higher Eukaryote and Prokaryotes Nucleotide-binding (HEPN) domain.
3. The composition of claim 1 , wherein at least the one or more nucleic acid components is engineered for use in modifying the mammalian target locus of interest comprising the target RNA sequence.
4. The composition of claim 1 , wherein at least one of said one or more localization signals is a nuclear localization signal (NLS) or a nuclear export signal (NES).
5. The composition of claim 1 , 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).
6. The composition of claim 5 , wherein the one or more regulatory elements comprise one or more promoters or one or more inducible promoters.
7. The composition of claim 1 , wherein the Cas13a CRISPR-Cas effector protein is associated with one or more functional domains.
8. The composition of claim 1 , wherein the effector protein contains one or more mutations within a HEPN Domain corresponding to R597A, H602A, R1278A, and/or H1283A of Leptotrichia shahii , or the corresponding amino acids in a Cas13a ortholog.
9. The composition of claim 1 , wherein the nucleic acid component comprises a dual direct repeat sequence.
10. The composition of claim 1 , wherein the one or more polynucleotide molecules are comprised within one or more vectors.
11. The composition of claim 10 , wherein the one or more vectors are viral vectors.
12. The composition of claim 11 , wherein the one or more viral vectors comprise one or more retroviral, lentiviral, adenoviral, adeno-associated or herpes simplex viral vectors.
13. The composition of claim 1 , wherein the assembled complex is comprised in a delivery system.
14. The composition of claim 1 , wherein the non-naturally occurring or engineered composition is delivered via a delivery vehicle comprising liposome(s), particle(s), exosome(s), microvesicle(s), a gene-gun or one or more viral vector(s).
15. A eukaryotic cell engineered to comprise or express, the composition or a component thereof of:
a non-naturally occurring or engineered composition comprising
i) a Cas13a CRISPR-Cas effector protein or one or more polynucleotide molecules encoding Cas13a CRISPR-Cas effector protein; and
ii) one or more nucleic acid components, the one or more nucleic acid components comprising a heterologous guide sequence that is capable of hybridizing to a target RNA sequence, whereby the effector protein forms a complex with the one or more nucleic acid components, the one or more nucleic acid components directs the complex to the target RNA sequence in a mammalian target locus and the complex binds to the target RNA sequence;
wherein the Cas13a CRISPR-Cas effector protein is selected from Cas13a CRISPR-Cas proteins obtained from Eubacteriaceae bacterium CHKCI004 , Blautia sp. Marseille-P2398, RNA-binding protein S1 Chloroflexus aggregans, Demequina aurantiaca, Thalassospira sp. TSL5-1, SAMN04487830_13920 Pseudobutyrivibrio sp. OR37, SAMN02910398_00008 Butyrivibrio sp. YAB3001 , Leptotrichia sp. Marseille-P3007 , Bacteroides ihuae , SAMN05216357_1045 Porphyromonadaceae bacterium KH3CP3RA, Listeria riparia , and Insolitispirillum peregrinum.
16. The cell according to claim 15 , wherein the cell comprises a mammalian cell.
17. A multicellular organism comprising one or more cells according to claim 15 .
18. A plant or animal model comprising one or more cells according to claim 15 , wherein said one or more cells express the composition or a component thereof.
19. A method of modifying a target sequence, the method comprising delivering to a locus of interest comprising the mammalian target sequence a non-naturally occurring or engineered composition comprising i) a Cas13a CRISPR-Cas effector protein fused to one or more localization signals, or one or more polynucleotide molecules encoding a Cas13a CRISPR-Cas effector protein fused to one or more localization signals; and
ii) one or more nucleic acid components, the one or more nucleic acid components comprising a heterologous guide sequence that is capable of hybridizing to a target RNA sequence, whereby the effector protein forms a complex with the one or more nucleic acid components, the one or more nucleic acid components directs the complex to the target RNA sequence in a mammalian target locus and the complex binds to the target RNA sequence;
wherein the Cas13a CRISPR-Cas effector protein is selected from Cas13a CRISPR-Cas proteins obtained from Eubacteriaceae bacterium CHKCI004 , Blautia sp. Marseille-P2398, RNA-binding protein S1 Chloroflexus aggregans, Demequina aurantiaca, Thalassospira sp. TSL5-1, SAMN04487830_13920 Pseudobutyrivibrio sp. OR37, SAMN02910398_00008 Butyrivibrio sp. YAB3001 , Leptotrichia sp. Marseille-P3007, Bacteroides ihuae , SAMN05216357_1045 Porphyromonadaceae bacterium KH3CP3RA, Listeria riparia, Insolitispirillum peregrinum, Herbinix hemicellulosilityca , and Leptrotrichia buccalis C-1013-b.
20. The method of claim 19 , wherein the modification of the target sequence comprises a strand break.
21. The method of claim 19 , wherein the Cas13a CRISPR-Cas effector protein is associated with one or more functional domains.
22. The method of claim 21 , wherein the one or more functional domains modifies transcription or translation of the target locus.
23. The method of claim 21 , wherein the effector protein contains one or more mutations within an HEPN Domain corresponding to R597A, H602A, R1278A, and/or H1283A H1283A of Leptotrichia shahii , or the corresponding amino acids in a Cas13a ortholog, whereby the complex can deliver an epigenetic modifier or a transcriptional or translational activation or repression signal.
24. The method of claim 19 , wherein at least one of said one or more said localization signals is a nuclear localization signal (NLS) or a nuclear export signal (NES).
25. The method of claim 19 , 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).
26. The composition of claim 25 , wherein the one or more regulatory elements comprise one or more promoters or one or more inducible promoters.