CRISPR enzymes and systems

Patent metadata

Field	Value
Publication number	US-9790490-B2
Application number	US-201514975085-A
Country	US
Kind code	B2
Filing date	Dec 18, 2015
Priority date	Jun 18, 2015
Publication date	Oct 17, 2017
Grant date	Oct 17, 2017

Abstract

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.

First claim

What is claimed:

1. An engineered, non-naturally occurring system comprising
a) a Cpf1 effector protein, and
b) at least one engineered guide polynucleotide designed to form a complex with the Cpf1effector protein and comprising a guide sequence, wherein the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell; and
wherein 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.

2. An engineered, non-naturally occurring system comprising
a) one or more nucleotide sequences encoding a Cpf1 effector protein, and
b) one or more nucleotide sequences encoding at least one engineered guide polynucleotide designed to form a complex with the Cpf1 effector protein and comprising a guide sequence, wherein the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell; and
wherein 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.

3. An engineered, non-naturally occurring vector system comprising one or more vectors comprising
a) a first regulatory element operably linked to one or more nucleotide sequences encoding a Cpf1 effector protein;
b) a second regulatory element operably linked to one or more nucleotide sequences encoding at least one engineered guide polynucleotide designed to form a complex with the Cpf1 effector protein and comprising a guide sequence, wherein the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell; and
wherein the system lacks a tracr sequence, components (a) and (b) are located on the same or different vectors of the system, 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.

4. An engineered, non-naturally occurring system comprising
a) a Cpf1 effector protein, or one or more nucleotide sequences encoding the Cpf1 effector protein, and
b) at least one engineered guide polynucleotide designed to form a complex with the Cpf1 effector protein and comprising a guide sequence, wherein the guide sequence is designed to hybridize with a target sequence in a eukaryotic cell, or one or more nucleotide sequences encoding the at least one engineered guide polynucleotide;
wherein 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.

5. The system according to claim 4 , comprising the at least one engineered guide polynucleotide.

6. The system according to claim 4 , comprising the one or more nucleotide sequences encoding the Cpf1 effector protein.

7. The system according to claim 4 , comprising the Cpf1 effector protein.

8. The system according to claim 4 , comprising the one or more nucleotide sequences encoding the at least one engineered guide polynucleotide.

9. The system according to claim 1 , 2 , 3 , or 4 , wherein the complex of the at least one engineered guide polynucleotide and the Cpf1 effector protein causes cleavage distally of the target sequence.

10. The system according to claim 9 , wherein said cleavage generates a staggered double stranded break with a 4- or 5-nt 5′ overhang.

11. The system according to claim 1 , 2 , 3 , or 4 , wherein said target sequence is 3′ of a Protospacer Adjacent Motif (PAM) and the PAM comprises a 5′ T-rich motif.

12. The system according to claim 1 , 2 , 3 , or 4 , wherein the Cpf1 effector protein is of a bacterial species listed in FIG. 64A or FIG. 64B .

13. The system according to claim 12 , wherein the bacterial species is selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10 , Parcubacteria bacterium GW2011_GWC2_44_17 , Smithella sp. SCADC, Acidaminococcus sp. BV3L6 , Lachnospiraceae bacterium MA2020 , Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237 , Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae.

14. The system according to claim 1 , 2 , 3 , or 4 , wherein said target sequence is 3′ of a Protospacer Adjacent Motif (PAM) and the PAM sequence is TTN, where N is A/C/G or T and the Cpf1 effector protein is FnCpf1, or the PAM sequence is TTTV, where V is A/C or G and the Cpf1 effector protein is PaCpf1p, LbCpf1 or AsCpf1.

15. The system according to claim 1 , 2 , 3 , or 4 , wherein the Cpf1 effector protein comprises one or more nuclear localization signals.

16. The system according to claim 2 , 3 , or 4 , wherein the one or more nucleotide sequences encoding the Cpf1 effector protein is/are codon optimized for expression in a eukaryotic cell.

17. The system according to claim 2 , 3 , or 4 , wherein components (a) and (b) are on one vector.

18. The system according to claim 2 or 4 , wherein the one or more nucleotide sequences of components (a) and (b) are a single nucleic acid molecule.

19. The system according to claim 1 , 2 , 3 , or 4 , wherein the eukaryotic cell is an animal or human cell.

20. The system according to claim 1 , 2 , 3 , or 4 , wherein the eukaryotic cell is a plant cell.

21. The system according to claim 1 , 2 , 3 , or 4 , wherein the guide sequence is linked to a direct repeat sequence.

22. The system according to claim 1 , 2 , 3 , or 4 , wherein the complex, engineered guide polynucleotide or Cpf1 effector protein is conjugated to at least one sugar moiety.

23. The system according to claim 22 , wherein the sugar moiety comprises N-acetyl galactosamine (GalNAc) or triantennary GalNAc.

24. The system according to claim 1 , 2 , 3 , or 4 , wherein the engineered guide polynucleotide comprises at least one modified nucleotide.

25. A delivery particle comprising the system according to claim 1 , 2 , 3 , or 4 .

26. The delivery particle of claim 25 , wherein the Cpf1 effector protein is complexed with the engineered guide polynucleotide.

27. The delivery particle of claim 25 , further comprising a lipid, a sugar, a metal or a protein.

28. The delivery particle of claim 26 , further comprising a lipid, a sugar, a metal or a protein.

29. A method of modifying a locus of interest having a target sequence of a eukaryotic cell comprising delivering the system according to claim 1 , 2 , 3 , or 4 , to said locus of interest, wherein the guide sequence hybridizes with the target sequence whereby modification of the locus of interest occurs.

30. The method of claim 29 , wherein the locus of interest is within a eukaryotic cell.

31. The method of claim 30 , wherein the cell is an animal cell.

32. The method of claim 30 , wherein the cell is an animal or human cell.

33. The method of claim 30 , wherein the cell

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College
• Broad Inst Inc
• Massachusetts Inst Technology

Inventors

• Zhang Feng
• Zetsche Bernd
• Slaymaker Ian
• Gootenberg Jonathan
• Abudayyeh Omar O

Classifications

• C12N2310/3519

  Fusion with another nucleic acid · CPC title

• C12N15/10 Primary

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12N15/11 Primary

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N15/8201

  Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation · CPC title

• C12N2310/16

  Aptamers · CPC title