Crispr enzymes and systems

Patent metadata

Field	Value
Publication number	US-11634755-B2
Application number	US-202117554333-A
Country	US
Kind code	B2
Filing date	Dec 17, 2021
Priority date	Jun 18, 2015
Publication date	Apr 25, 2023
Grant date	Apr 25, 2023

Abstract

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.

First claim

What is claimed:

1. A composition comprising, (a) a Cas polypeptide that comprises a RuvC-like nuclease domain and a zinc finger, but does not comprise an HNH domain, or a polynucleotide encoding the Cas polypeptide, and (b) one or more engineered nucleic acid components lacking a tracr sequence that form a CRISPR-Cas complex with the Cas polypeptide and that is capable of directing sequence-specific binding of said complex to a target polynucleotide sequence adjacent to a protospacer adjacent motif (PAM) in the genome of a eukaryotic cell, or a polynucleotide encoding the one or more engineered nucleic acid components.

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

3. The composition of claim 1 , that is capable of directing cleavage at the target sequence of the polynucleotide.

4. The composition of claim 1 , wherein the one or more nucleic acid components comprises a guide RNA.

5. The composition of claim 1 , wherein the Cas polypeptide comprises one or more nuclear localization signals and/or the one or more nucleic acid components comprise one or more modified nucleotides or one or more non-nucleotide moieties.

6. The composition of claim 1 , wherein the Cas polypeptide is linked to a heterologous functional domain.

7. The composition of claim 1 , wherein the composition comprises the formed CRISPR-Cas complex.

8. The composition of claim 1 , wherein the composition comprises one or more vectors encoding the Cas polypeptide and the one or more nucleic acid components.

9. The composition of claim 8 , wherein a polynucleotide sequence in the one or more vectors encoding the Cas polypeptide is codon optimized for expression in a eukaryotic cell.

10. The composition of claim 8 , wherein the one or more vectors are viral vectors, optionally an adenoviral vector, a lentiviral vector, or an adeno-associated viral vector.

11. The composition of claim 1 , wherein the composition comprises a mRNA encoding the Cas polypeptide.

12. The composition of claim 11 , wherein the mRNA encoding the Cas polypeptide is comprised in a lipid nanoparticle, a liposome, an exosome, or a microvesicle.

13. A method of targeting a polynucleotide, comprising contacting a sample comprising the polynucleotide with the composition of claim 1 .

14. The method of claim 13 , wherein the Cas polypeptide is a Type V Cas polypeptide.

15. The method of claim 13 , wherein the target sequence is in a eukaryotic cell, and wherein contacting the eukaryotic cell with the CRISPR-Cas complex results in modification of a gene or gene product, or modification in the expression of a gene product.

16. The method of claim 13 , wherein the target sequence is in a eukaryotic cell, and wherein contacting the eukaryotic cell with the CRISRP-Cas complex results in cleavage of the target sequence.

17. A composition comprising (a) a Cas polypeptide that comprises a RuvC-like nuclease domain, but does not comprise an HNH domain, or a polynucleotide encoding the Cas polypeptide, and (b) one or more engineered nucleic acid components lacking a tracr sequence that form a complex with the Cas polypeptide and that is capable of directing sequence-specific binding of said complex to a target polynucleotide sequence adjacent to a protospacer adjacent motif (PAM) in the genome of a eukaryotic cell, or a polynucleotide encoding the one or more engineered nucleic acid components, wherein the Cas polypeptide comprises at least one mutation in the RuvC-like nuclease domain and is catalytically inactive.

18. The composition of claim 17 , wherein the Cas polypeptide is a Type V Cas polypeptide.

19. The composition of claim 17 , wherein the as polypeptide comprises one or more nuclear localization signals and/or one or more nucleic acid components comprising one or more modified nucleotides or one or more non-nucleotide moieties.

20. The composition of claim 17 , wherein the Cas polypeptide is linked to a heterologous functional domain.

21. The composition of claim 17 , wherein the composition comprises the formed CRISPR-Cas complex.

22. The composition of claim 17 , wherein the composition comprises one or more vectors encoding the Cas polypeptide and the one or more nucleic acid components.

23. The composition of claim 22 , wherein a polynucleotide sequence in the one or more vectors encoding the Cas polypeptide is codon optimized for expression in a eukaryotic cell.

24. The composition of claim 22 , wherein the one or more vectors are viral vectors, optionally an adenoviral vector, a lentiviral vector, or an adeno-associated viral vector.

25. The composition of claim 17 , wherein the composition comprises a mRNA encoding the Cas polypeptide.

26. The composition of claim 25 , wherein the mRNA encoding the Cas polypeptide is comprised in a lipid nanoparticle, a liposome, an exosome, or a microvesicle.

27. A method of targeting a polynucleotide, comprising contacting a sample comprising the polynucleotide with the composition of claim 17 .

28. The method of claim 27 , wherein the Cas polypeptide is a Type V Cas polypeptide.

29. The method of claim 27 , wherein the target sequence is in a eukaryotic cell.

30. The method of claim 29 , wherein contacting the eukaryotic cell with the CRISPR-Cas complex results in modification of a gene or gene product, or modification in the expression of a gene product.

Assignees

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

Inventors

• Zhang Feng
• Zetsche Bernd
• Gootenberg Johnathan S
• Abudayyeh Omar O
• Slaymaker Ian

Classifications

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2310/20

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

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12Q1/6832 Primary

  Enhancement of hybridisation reaction · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title