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 method of targeting a polynucleotide, comprising: contacting a sample that comprises the polynucleotide with a CRISPR-Cas complex comprising (a) a Cas protein that does not comprise an HNH domain or a polynucleotide encoding the Cas protein and (b) an engineered guide without a tracrRNA that is capable of directing sequence-specific binding of the complex to a target sequence of the polynucleotide or a polynucleotide encoding the engineered guide.
2. The method of claim 1 , further comprising detecting binding of the complex to the polynucleotide.
3. The method of claim 1 , wherein the Cas protein comprises a contiguous RuvC-like domain.
4. The method of claim 1 , wherein the Cas protein is a Type V Cas protein.
5. The method of claim 4 , wherein the Type V Cas protein is a Cpf1.
6. The method of claim 1 , wherein the Cas protein is fused to one or more heterologous functional domains.
7. The method of claim 6 , wherein the one or more heterologous functional 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.
8. The method of claim 6 , 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.
9. The method of claim 6 , wherein the one or more heterologous functional domains comprise a deaminase domain.
10. The method of claim 1 , wherein the Cas protein comprises at least one mutation.
11. The method of claim 1 , wherein the Cas protein is obtained from a bacterial species selected from the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Campylobacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium , and Acidaminococcus.
12. The method of claim 1 , wherein the complex is formed outside a prokaryotic cell.
13. The method of claim 1 , wherein the target sequence is in a eukaryotic cell.
14. The method of claim 13 , wherein a gene product is modified or the amount or expression of a gene product is modified.
15. The method of claim 13 , wherein the target sequence is in a promoter or enhancer.
16. The method of claim 13 , further comprising delivering an exogenous polynucleotide template to the eukaryotic cell for targeted integration.
17. The method of claim 1 , wherein the Cas protein is fused to at least one heterologous nuclear localization signal.
18. The method of claim 1 , wherein the engineered guide comprises at least one chemical modification comprising 2′-O-methyl, 2′-O-methyl 3′ phosphorothioate, or 2′-O-methyl 3′ thioPACE.
19. A method of modifying a target locus of interest comprising: delivering to said locus a non-naturally occurring or engineered composition comprising (a) a Type V CRISPR-Cas loci effector protein or a polynucleotide encoding the effector protein and (b) a single nucleic acid component comprising a guide sequence linked to a direct repeat sequence or a polynucleotide encoding the single nucleic acid component, wherein the effector protein forms a complex with the single nucleic acid component and upon binding of the complex to the target locus of interest the effector protein induces a modification of the target locus of interest.
20. The method of claim 19 , wherein the effector protein comprises a contiguous RuvC-like domain.
21. The claim 19 , wherein the effector protein does not comprise an HNH domain.
22. The method of claim 19 , wherein the effector protein is a Cpf1.
23. The method of claim 19 , wherein the effector protein is obtained from a bacterial species selected from the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Campylobacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium , and Acidaminococcus.
24. The method of claim 19 , wherein the effector protein is fused to one or more heterologous functional domains.
25. The method of claim 24 , wherein the one or more heterologous functional 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.
26. The method of claim 24 , 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.
27. The method of claim 24 , wherein the one or more heterologous functional domains comprise a deaminase domain.
28. The method of claim 19 , wherein the effector protein comprises at least one mutation.
29. The method of claim 19 , wherein the target locus of interest is a disease-associated DNA.
30. The method of claim 19 , wherein the target locus of interest is in a eukaryotic cell.
31. The method of claim 30 , wherein the target locus of interest is in a promoter or enhancer.
32. The method of claim 30 , further comprising delivering an exogenous polynucleotide template to the eukaryotic cell for targeted integration.
33. The method of claim 19 , further comprising detecting binding of the complex to the target locus of interest.
34. The method of claim 19 , wherein the effector protein is fused to at least one heterologous nuclear localization signal.
35. The method of claim 19 , wherein the single nucleic acid component comprises at least one chemical modification comprising 2′-O-methyl, 2′-O-methyl 3′ phosphorothioate, or 2′-O-methyl 3′ thioPACE.