CRISPR enzymes and systems

Patent metadata

Field	Value
Publication number	US-11180751-B2
Application number	US-201715842073-A
Country	US
Kind code	B2
Filing date	Dec 14, 2017
Priority date	Jun 18, 2015
Publication date	Nov 23, 2021
Grant date	Nov 23, 2021

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 is:

1. A Type V non-naturally occurring, engineered composition comprising
a) a Type V Cas polypeptide comprising one or more catalytic motifs of a RuvC nuclease domain but not comprising a HNH domain, and one or more nucleic acid components comprising a heterologous guide sequence and a tracrRNA sequence that are capable of forming a complex with the Type V Cas polypeptide and directing site-specific binding of said complex to a target sequence; or
b) a Type V Cas polypeptide comprising one or more catalytic motifs of the RuvC nuclease domain and a Zn finger region with at least one Zn-binding cysteine, but not comprising a HNH domain, and one or more nucleic acid components comprising a heterologous guide sequence that is capable of forming a complex with the Type V Cas polypeptide and directing sequence-specific binding of said complex to a target sequence.

2. The composition of claim 1 , wherein the Type V Cas polypeptide comprises an arginine rich cluster, but which does not bind Zinc.

3. The composition of claim 1 , wherein the Type V Cas polypeptide is a C2c1 or C2c3 polypeptide.

4. The composition of claim 3 , wherein the C2c1 polypeptide is obtained from a bacterial species selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus contaminans, Desulfovibrio inopinatus, Desulfonatronum thiodismutans, Opitutaceae bacterium TAV5 , Tuberibacillus calidus, Bacillus thermoamylovorans, Brevibacillus sp. CF112, Bacillus sp. NSP2.1 , Desulfatirhabdium butyrativorans, Alicyclobacillus herbarius, Citrobacter freundii, Brevibacillus agri , and Methylobacterium nodulans.

5. The composition of claim 3 , wherein the C2c1 polypeptide comprises at least 95% sequence identity to a C2c1 polypeptide selected from the group consisting of: SEQ ID NOs: 548-567.

6. The composition of claim 3 , wherein the Type V Cas polypeptide of a) is C2c1 polypeptide, or the Type V Cas polypeptide of b) is C2c3 polypeptide.

7. The composition according to claim 1 , wherein the Type V Cas polypeptide comprises one or more nuclear localization signals.

8. The composition according to claim 1 , wherein the Type V Cas polypeptide comprises a mutation of one or more amino acid residues in a catalytically active domain of the effector protein and has reduced or abolished nuclease activity compared with a Type V Cas polypeptide lacking said one or more mutations.

9. The composition according to claim 1 , wherein the Type V Cas polypeptide is linked to a heterologous functional domain.

10. A vector system comprising one or more vectors, the one or more vectors comprising one or more polynucleotide molecules encoding components of the non-naturally occurring or engineered composition of claim 1 .

11. The vector system according to claim 10 , wherein the one or more polynucleotide molecules comprise one or more regulatory elements capable of expressing the polypeptide(s) and/or the nucleic acid component(s), optionally wherein the one or more regulatory elements comprise inducible promotors.

12. The vector system according to claim 10 , wherein the Type V Cas polypeptide is codon optimized for expression in a eukaryotic cell.

13. The method of claim 12 , wherein modifying comprises cleaving the sequence.

14. The method of claim 12 , wherein the target sequence comprises DNA.

15. An isolated eukaryotic or prokaryotic cell transiently or non-transiently transfected with the vector of claim 10 .

16. An in vitro, in vivo, or ex vivo method of targeting a target sequence in a eukaryotic or prokaryotic cell, the method comprising: contacting a sample with the engineered composition of claim 1 .

17. The method of claim 16 , wherein the sample comprises a polynucleotide comprising the target sequence in a eukaryotic cell.

18. The method of claim 16 , wherein the Type V Cas polypeptide and nucleic acid component(s) are provided via one or more polynucleotide molecules encoding the polypeptide(s) and/or the nucleic acid component(s), and wherein the one or more polynucleotide molecules are capable of expressing the polypeptide(s) and/or the nucleic acid component(s).

19. The method of claim 16 , wherein the engineered composition is delivered via liposomes, nanoparticles, exosomes, microvesicles, a gene-gun or one or more viral vectors.

20. The method of claim 16 , further comprising modifying said target sequence.

Assignees

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

Inventors

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

Classifications

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• A61K48/005

  characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N15/11

  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

• C12N15/111

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