CRISPR enzymes and systems

Patent metadata

Field	Value
Publication number	US-12264359-B2
Application number	US-202016909101-A
Country	US
Kind code	B2
Filing date	Jun 23, 2020
Priority date	Jun 18, 2015
Publication date	Apr 1, 2025
Grant date	Apr 1, 2025

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 is:

1. A method of altering expression of a gene in a mammalian cell, comprising delivering to the mammalian cell:
(a) a Cpf1 protein, or a polynucleotide encoding the Cpf1 protein, and
(b) a Cpf1 guide comprising a guide sequence capable of hybridizing with a target sequence associated with the gene, or a polynucleotide encoding the Cpf1 guide,
wherein a CRISPR complex formed by the Cpf1 guide and the Cpf1 protein binds to the target sequence and alters expression of the gene, thereby producing a modified mammalian cell.

2. The method of claim 1 , wherein the delivering step comprises delivering to the mammalian cell the Cpf1 guide and the Cpf1 protein.

3. The method of claim 2 , wherein the delivering step comprises delivering to the mammalian cell a CRISPR complex formed by the Cpf1 guide and the Cpf1 protein.

4. The method of claim 1 , wherein the delivering step comprises delivering to the mammalian cell a vector encoding the Cpf1 protein, a vector encoding the Cpf1 guide, or a vector encoding both the Cpf1 protein and the Cpf1 guide.

5. The method of claim 4 , wherein the vector is a viral vector.

6. The method of claim 5 , wherein the viral vector is an adenoviral vector, a lentiviral vector, or an adeno-associated viral vector.

7. The method of claim 1 , wherein the delivering step comprises delivering an mRNA encoding the Cpf1 protein to the mammalian cell.

8. The method of claim 7 , wherein the mRNA encoding the Cpf1 protein is comprised in a lipid nanoparticle, a liposome, an exosome, or a microvesicle.

9. The method of claim 1 , wherein (a) and (b) are delivered to the mammalian cell simultaneously.

10. The method of claim 1 , wherein (a) and (b) are delivered to the mammalian cell sequentially.

11. The method of claim 1 wherein (a) and (b) are delivered to the mammalian cell ex vivo.

12. The method of claim 1 , wherein (a) and (b) are delivered to the mammalian cell in vivo.

13. The method of claim 1 , wherein the delivering step comprises microinjection, electroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions.

14. The method of claim 1 , wherein the mammalian cell is a rodent cell, an ungulate cell, or a primate cell.

15. The method of claim 1 , wherein the mammalian cell is a human cell.

16. The method of claim 15 , wherein the mammalian cell is a hematopoietic cell or a lymphocyte.

17. The method of claim 15 , wherein the mammalian cell is a hematopoietic CD34+ stem/progenitor cell, a natural killer cell, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a tumor-infiltrating lymphocyte.

18. The method of claim 1 , wherein the gene is associated with a genetic disease or disorder.

19. The method of claim 18 , wherein the genetic disease or disorder is a blood disease or disorder.

20. The method of claim 19 , wherein the genetic disease or disorder is Sickle Cell Anemia, Beta-Thalassemia, or Hemophilia.

21. The method of claim 18 , wherein the genetic disease or disorder is an ophthalmic or ocular disease or disorder.

22. The method of claim 21 , wherein the genetic disease or disorder is Leber Congenital Amaurosis, Usher Syndrome, Retinitis Pigmentosa, or Primary Open Angle Glaucoma.

23. The method of claim 18 , wherein the genetic disease or disorder is a muscle disease or disorder.

24. The method of claim 23 , wherein the genetic disease or disorder is Cystic Fibrosis or Duchenne Muscular Dystrophy.

25. The method of claim 18 , wherein the genetic disease or disorder is a cancer.

26. The method of claim 25 , wherein the modified mammalian cell is a modified lymphocyte, and wherein the method further comprises administering the modified lymphocyte to a cancer patient.

27. The method of claim 26 , wherein the modified lymphocyte has altered expression of one or more of PD1, CTLA4, TRAC, TRBC, B2M, and MHC2TA.

28. The method of claim 1 , wherein the Cpf1 protein is from 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 , or Porphyromonas macacae.

29. The method of claim 1 , wherein the Cpf1 protein is Francisella novicida U112 Cpf1 (FnCpf1), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006 (LbCpf1).

30. The method of claim 1 , wherein the Cpf1 protein comprises at least one nuclear localization sequence.

31. The method of claim 1 , wherein the Cpf1 protein comprises at least one mutation in a catalytic domain and is catalytically inactive.

32. The method of claim 1 , wherein the Cpf1 protein is linked to a heterologous functional domain.

33. The method of claim 32 , wherein the heterologous functional domain has one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, and nucleic acid binding activity.

34. The method of claim 1 , wherein the Cpf1 guide comprises the guide sequence linked to a direct repeat sequence.

35. The method of claim 1 , wherein the Cpf1 guide comprises RNA nucleotides.

36. The method of claim 1 , wherein the Cpf1 guide comprises one or more modified nucleotides or one or more non-nucleotide moieties.

37. The method of claim 1 , wherein the Cpf1 guide comprises at least one chemical modification, wherein the chemical modification is a methylene bridge between carbon atoms of a ribose ring, 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.

38. The method of claim 1 , wherein the target sequence is adjacent to a T-rich protospacer adjacent motif (PAM).

39. The method of claim 38 , wherein the Cpf1 protein is FnCpf1 and the PAM is TTN, where Nis A, C, G or T; or wherein the Cpf1 protein is AsCpf1 or LbCpf1 and the PAM sequence is TTTV, where Vis A, C or G.

40. The method of claim 1 , wherein the CRISPR complex introduces a strand break at the target sequence.

41. The method of claim 40 , wherein the CRISPR complex introduces a staggered double-stranded break with a 5′ overhang.

42. The method of claim 40 , wherein the CRISPR complex introduces a staggered double-stranded break with a 4-nt

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College

Inventors

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

Classifications

• C12Q1/6816

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

• C12N9/22 Primary

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

• C12N2310/20

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

• C12N2800/80

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