CRISPR enzymes and systems

Patent metadata

Field	Value
Publication number	US-11060115-B2
Application number	US-201715482603-A
Country	US
Kind code	B2
Filing date	Apr 7, 2017
Priority date	Jun 18, 2015
Publication date	Jul 13, 2021
Grant date	Jul 13, 2021

Abstract

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.

First claim

What is claimed:

1. An engineered composition formulated for use in eukaryotic cells comprising
i) a Cas polypeptide, or a polynucleotide encoding the Cas polypeptide, the Cas polypeptide comprising two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains and comprising 95% sequence identity to any one of the polypeptides of SEQ ID NO: 573-591, wherein the Cas polypeptide comprises one or more nuclear localization sequences and the polynucleotide encoding the Cas polypeptide is optionally codon optimized for expression in eukaryotic cells; and
ii) one or more nucleic acid components, or a polynucleotide encoding the one or more nucleic acid components, wherein the one or more nucleic acid components is capable of forming a CRISPR-Cas complex with the Cas polypeptide, and wherein said one or more nucleic acid components can direct sequence specific binding of the complex to a target sequence of a RNA polynucleotide.

2. The composition of claim 1 , wherein the Cas polypeptide is a Type VI Cas polypeptide.

3. An engineered composition comprising
i) a Cas polypeptide, or a polynucleotide encoding the Cas polypeptide, the Cas polypeptide comprising 95% sequence identity to any one of the polypeptides of SEQ ID NO: 573-587 and 591, and comprising one or more mutations that abrogate nuclease activity, the one or more mutations corresponding to substitutions in the polypeptide of SEQ ID NO: 591 selected from the group consisting of R597A, H602A, R1278A and H1283A; and
ii) one or more nucleic acid components, or a polynucleotide encoding the one or more nucleic acid components, wherein the one or more nucleic acid components is capable of forming a CRISPR-Cas complex with the Cas polypeptide, and wherein said one or more nucleic acid components can direct sequence specific binding of the complex to a target sequence of a RNA polynucleotide.

4. The composition of claim 1 , wherein the Cas polypeptide is 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, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifactor, Mycoplasma, Camplyobacter, Leptotrichia, Rhodobacter, Lachnospiraceae, Carnobacterium , and Paludibacter.

5. The composition of claim 1 , wherein the one or more nucleic acid components comprise a dual direct repeat sequence.

6. The composition of claim 1 , wherein the polynucleotide encoding the Cas polypeptide and/or the polynucleotide encoding the one or more nucleic acid components are operably configured to express the polypeptide and/or the one or more nucleic acid components.

7. The composition of claim 6 , wherein the polynucleotide encoding the Cas polypeptide and/or the polynucleotide encoding the one or more nucleic acid components comprise one or more regulatory elements operably configured to express the polypeptide and/or the one or more nucleic acid components, and wherein the one or more regulatory elements optionally comprise promoters or inducible promoters.

8. The composition of claim 3 , wherein the complex delivers an epigenetic modifier, or a transcriptional or translational activation or repression signal.

9. The composition of claim 3 , wherein the complex delivers a functional domain that modifies transcription or translation of the target sequence.

10. The composition of claim 1 , wherein the polynucleotide encoding the Cas polypeptide and the polynucleotide encoding the one or more nucleic acid components are comprised within one or more vectors.

11. The composition of claim 10 , wherein the one or more vectors are viral vectors are selected from retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

12. The composition of claim 1 , wherein the polynucleotide molecules encoding the Cas polypeptide and/or the one or more nucleic acid components are comprised in a delivery system.

13. The composition of claim 12 , wherein the polynucleotide molecules encoding the Cas polypeptide and the one or more nucleic acid components comprise one or more regulatory elements operably configured to express the polypeptide and/or the one or more nucleic acid components, and the one or more regulatory elements comprise a promoter or an inducible promoter.

14. The composition of claim 1 , wherein the complex or one or more of its components is comprised in a delivery system.

15. The composition of claim 3 , wherein the Cas polypeptide is a Type VI Cas polypeptide.

16. The composition of claim 3 , wherein the Cas polypeptide is 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, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifactor, Mycoplasma, Camplyobacter, Leptotrichia, Rhodobacter, Lachnospiraceae, Carnobacterium , and Paludibacter.

17. The composition of claim 3 , wherein the Cas polypeptide comprises at least one or more nuclear localization signals.

18. The composition of claim 3 , wherein the one or more nucleic acid components comprise a dual direct repeat sequence.

19. The composition of claim 3 , wherein the polynucleotide encoding the Cas polypeptide and/or the polynucleotide encoding the one or more nucleic acid components are operably configured to express the polypeptide and/or the nucleic acid components.

20. The composition of claim 3 , wherein the polynucleotide encoding the Cas polypeptide and/or the polynucleotide encoding the one or more nucleic acid components are comprised within one or more vectors.

21. The composition of claim 20 , wherein the one or more vectors are viral vectors are selected from retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

22. The composition of claim 21 , wherein the polynucleotide molecules encoding the Cas polypeptide and/or the one or more nucleic acid components are comprised in a delivery system.

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College
• Univ Rutgers
• Skolkovo Institute Of Science And Tech
• Us Health

Inventors

• Severinov Konstantin
• Zhang Feng
• Wolf Yuri I
• Shmakov Sergey
• Semenova Ekaterina
• Minakhin Leonid
• Makarova Kira S
• Koonin Eugene
• Konermann Silvana
• Joung Julia
• Gootenberg Jonathan S
• Abudayyeh Omar O
• Lander Eric S

Classifications

• C12N15/102 Primary

  Mutagenizing nucleic acids · 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

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N2310/111

  spanning the whole gene, or a large part of it · CPC title

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title