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 or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.
1. A method of modifying a eukaryotic target locus of interest comprising: delivering to said locus of interest or a cell containing the locus of interest a system comprising:
a) at least one Cpf1 effector protein, and
b) an engineered guide polynucleotide comprising a guide sequence, which engineered guide polynucleotide is designed to form a complex with the Cpf1 effector protein, wherein:
the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell; the system lacks a tracr sequence;
the engineered guide polynucleotide and Cpf1 effector protein do not naturally occur together; and
a complex of the engineered guide polynucleotide and Cpf1 effector protein does not naturally occur, wherein the target locus of interest is modified.
2. A method of modifying a eukaryotic target locus of interest comprising: delivering to said locus of interest or a cell containing the locus of interest a system comprising:
a) at least one nucleotide sequence encoding a Cpf1 effector protein, and
b) at least one nucleotide sequence encoding an engineered guide polynucleotide comprising a guide sequence, which engineered guide polynucleotide is designed to form a complex with the Cpf1 effector protein, wherein:
the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell; the system lacks a tracr sequence;
the engineered guide polynucleotide and Cpf1 effector protein do not naturally occur together; and
a complex of the engineered guide polynucleotide and Cpf1 effector protein does not naturally occur, wherein the target locus of interest is modified.
3. A method of modifying a eukaryotic target locus of interest comprising: delivering to said locus of interest or a cell containing the locus of interest a vector system comprising at least one vector and:
a) a first regulatory element operably linked to at least one nucleotide sequence encoding a Cpf1 effector protein; and
b) a second regulatory element operably linked to at least one nucleotide sequence encoding an engineered guide polynucleotide comprising a guide sequence, which engineered guide polynucleotide is designed to form a complex with the Cpf1 effector protein, wherein:
the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell; the system lacks a tracr sequence;
the engineered guide polynucleotide and Cpf1 effector protein do not naturally occur together; and
a complex of the engineered guide polynucleotide and Cpf1 effector protein does not naturally occur, wherein the target locus of interest is modified.
4. The method of claim 3 wherein components (a) and (b) are located on the same vector.
5. The method of claim 3 , wherein the vector system comprises at least one viral vector.
6. A method of modifying a eukaryotic target locus of interest comprising: delivering to said locus of interest or a cell containing the locus of interest a system comprising:
a) at least one Cpf1 effector protein, or at least one nucleotide sequence encoding the at least one Cpf1 effector protein, and
b) an engineered guide polynucleotide comprising a guide sequence, which engineered guide polynucleotide is designed to form a complex with the Cpf1 effector protein, or at least one nucleotide sequence encoding the engineered guide polynucleotide, wherein:
the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell; the system lacks a tracr sequence;
the engineered guide polynucleotide and Cpf1 effector protein do not naturally occur together; and
a complex of the engineered guide polynucleotide and Cpf1 effector protein does not naturally occur, wherein the target locus of interest is modified.
7. The method of claim 6 , herein the target locus of interest is within a cell.
8. The method of claim 7 , wherein the cell is a eukaryotic cell.
9. The method of claim 7 , wherein the cell is an animal or human cell.
10. The method of claim 7 , wherein the cell is a plant cell.
11. The method of claim 6 , wherein the target locus of interest is comprised in a DNA molecule in vitro.
12. The method of claim 6 , wherein the target locus of interest is within cells of a cell line or an organism.
13. The method of claim 6 , wherein the complex, the engineered guide polynucleotide or the Cpf1 effector protein is conjugated to at least one sugar moiety.
14. The method of claim 13 , wherein the sugar moiety comprises N-acetyl galactosamine (GalNAc).
15. The method of claim 13 , wherein the sugar moiety comprises triantennary GalNAc.
16. The method of claim 6 , wherein the target locus of interest comprises relaxed or supercoiled DNA.
17. The method of claim 6 , wherein a single nucleic acid component comprises component (a) and (b) of the system.
18. The method of claim 6 , wherein the guide sequence is linked to a direct repeat sequence.
19. The method of claim 6 , wherein the modifying of the target locus of interest comprises a strand break.
20. The method of claim 19 , wherein the strand break comprises a staggered DNA double stranded break with a 4 or 5-nt 5′ overhang.
21. The method of claim 19 , wherein the modifying of the target locus of interest comprises integration of a DNA insert into a staggered DNA double stranded break.
22. The method of claim 6 , wherein the Cpf1 effector protein comprises at least one nuclear localization signal(s) (NLS(s)).
23. The method of claim 6 , wherein components (a) and (b) comprise the at least one nucleotide sequence encoding the engineered guide polynucleotide and the at least one nucleotide sequence encoding the Cpf1 effector protein, within at least one vector.
24. The method of claim 6 , wherein the system includes at least one regulatory element operably linked to: the at least one nucleotide sequence encoding the engineered guide polynucleotide, or the at least one nucleotide sequence encoding the Cpf1 effector protein, or both the at least one nucleotide sequence encoding the engineered guide polynucleotide and the at least one nucleotide sequence encoding the Cpf1 effector protein.
25. The method of claim 24 wherein the at least one regulatory element comprises at least one inducible promoter.
26. The method of claim 6 , wherein components (a) and (b) comprise the at least one nucleotide sequence encoding the engineered guide polynucleotide and the at least one nucleotide sequence encoding the Cpf1 effector protein, within a delivery system.
27. The method of claim 26 , wherein the delivery system comprises particles, vesicles, or at least one viral vector.
28. The method of claim 27 , wherein the particles comprise a lipid, a sugar, a metal or a protein.
29. The method of claim 27 , wherein the vesicles comprise exosomes or liposomes.
30. The method of claim 27 or 5 , wherein the at least one viral vector comprise at least one of adenovirus, at least one lentivirus or at least one adeno-associated virus.
31. The method of any one of claims 1 - 6 , wherein the target locus of interest comprises DNA.
32. The method of any one of claims 1 - 6 wherein the engineered guide polynucleotide comprises RNA.
33. The method of claim 32 wherein the engineered guide polynucleotide comprises one or more modified nucleotides or one or more non-nucleotide moieties.
34. The method of claim 33 wherein the modification comprises a chemical modification or the one or more non-nucleotide moieties comprises a protein or functional domain.
35. The method of claim 34 wherein the chemical modification comprises a methylene bridge between carbon atoms of a ribose ring or a phosphorothioate linkage or incorporation of 2′-O-methyl, 2′-O-methyl 3′ phosphorothioate, or 2′-O-methyl 3′thioPACE at one or more terminal nucleotides.
36. The method of claim 32 wherein the engineered guide polynucleotide comprises one or more modified nucleotides or one or more non-RNA moieties.
37. The method of claim 36 wherein the modification comprises a chemical modification or the one or more non-RNA moieties comprises a protein or functional domain.
38. The method of any one of claims 1 - 6 wherein the engineered guide polynucleotide comprises one or more modified nucleotides or one or more non-nucleotide moieties.
39. The method of claim 38 wherein the modification comprises a chemical modification or the one or more non-nucleotide moieties comprises a protein or functional domain.
40. The method of claim 39 wherein the chemical modification comprises a methylene bridge between carbon atoms of a ribose ring or a phosphorothioate linkage or incorporation of 2′-O-methyl, 2′-O-methyl 3′ phosphorothioate, or 2′-O-methyl 3′thioPACE at one or more terminal nucleotides.
41. The method of any one of claims 1 - 6 further comprising introducing a DNA template to the target locus of interest or cell.