Granted Patent
B2
US 12,371,737 · App. 16/909,064 · Granted Jul 29, 2025
CRISPR enzymes and systems
Inventors:
Feng Zhang (Cambridge, MA); Bernd Zetsche (Cambridge, MA); Jonathan S. Gootenberg (Cambridge, MA); Omar O. Abudayyeh (Cambridge, MA); Ian Slaymaker (Cambridge, MA)
Assignees:
THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY; PRESIDENT AND FELLOWS OF HARVARD COLLEGE
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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.
Claims (42)
1. A composition comprising a CRISPR-Cas system, comprising:
(a) a Type-V Cas protein, or a polynucleotide encoding the Type-V Cas protein, and
(b) an engineered guide engineered to hybridize with a target sequence adjacent to a protospacer motif (PAM) in the genome of a eukaryotic cell, or a polynucleotide encoding the engineered guide, wherein the engineered guide is capable of forming a CRISPR complex with the Type-V Cas protein.
2. The composition of claim 1 , wherein the composition comprises the engineered guide and the Type-V Cas protein.
3. The composition of claim 1 , wherein the composition comprises a CRISPR complex formed by the engineered guide and the Type-V Cas protein.
4. The composition of claim 1 , wherein the composition comprises one or more vectors encoding the engineered guide and the Type-V Cas protein.
5. The composition of claim 4 , wherein the composition comprises one or more viral vectors encoding the engineered guide and the Type-V Cas protein.
6. The composition of claim 5 , wherein the viral vectors comprise an adenoviral vector, a lentiviral vector, or an adeno-associated viral vector.
7. The composition of claim 1 , wherein the composition comprises an mRNA encoding the Type-V Cas protein.
8. The composition of claim 7 , wherein the mRNA encoding the Type-V Cas protein is comprised in a lipid nanoparticle, a liposome, an exosome, or a microvesicle.
9. The composition of claim 1 , wherein the Type-V Cas protein is from Francisella tularensis 1 , Prevotella albensis , Lachnospiraceae bacterium MC20171 , 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.
10. The composition of claim 1 , wherein the Type-V Cas protein is Francisella novicida U112 Cpf1 (FnCpf1), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006 (LbCpf1).
11. The composition of claim 1 , wherein the Type-V Cas protein comprises at least one nuclear localization sequence.
12. The composition of claim 1 , wherein the Type-V Cas protein comprises at least one mutation in a catalytic domain and is catalytically inactive.
13. The composition of claim 1 , wherein the Type-V Cas protein is linked to a heterologous functional domain.
14. The composition of claim 13 , 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.
15. The composition of claim 1 , wherein the engineered guide comprises the guide sequence linked to a direct repeat sequence.
16. The composition of claim 1 , wherein the engineered guide comprises RNA nucleotides.
17. The composition of claim 1 , wherein the engineered guide comprises one or more modified nucleotides or one or more non-nucleotide moieties.
18. The composition of claim 1 , wherein the engineered 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.
19. The composition of claim 1 , wherein the target sequence is adjacent to a T-rich PAM.
20. The composition of claim 19 , wherein the Type-V Cas protein is FnCpf1 and the PAM is TTN, where N is A, C, G or T; or wherein the Type-V Cas protein is AsCpf1 or LbCpf1 and the PAM sequence is TTTV, where V is A, C or G.
21. The composition of claim 1 , wherein the CRISPR complex generates a strand break at the target sequence.
22. The composition of claim 21 , wherein the strand break is a staggered double-stranded break with a 5′ overhang.
23. The composition of claim 21 , wherein the strand break is a staggered double-stranded break with a 4-nt or 5-nt overhang.
24. The composition of claim 21 , wherein the composition further comprises a single-stranded oligodeoxynucleotide as a template for homology-directed repair of the double-stranded break.
25. The composition of claim 21 , wherein the composition further comprises a double-stranded oligodeoxynucleotide for insertion into the double-stranded break.
26. The composition of claim 1 , wherein the eukaryotic cell is mammalian cell or a plant cell.
27. The composition of claim 26 , wherein the mammalian cell is a rodent cell, an ungulate cell, or a primate cell.
28. The composition of claim 27 , wherein the mammalian cell is a human cell.
29. The composition of claim 28 , wherein the mammalian cell is a hematopoietic cell or a lymphocyte.
30. The composition of claim 29 , 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.
31. The composition of claim 1 , wherein the target sequence is associated with a genetic disease or disorder.
32. The composition of claim 31 , wherein the target sequence is associated with a blood disease or disorder.
33. The composition of claim 32 , wherein the target sequence is associated with Sickle Cell Anemia, Beta-Thalassemia, or Hemophilia.
34. The composition of claim 31 , wherein the target sequence is associated with an ophthalmic or ocular disease or disorder.
35. The composition of claim 34 , wherein the target sequence is associated with Leber Congenital Amaurosis, Usher Syndrome, Retinitis Pigmentosa, or Primary Open Angle Glaucoma.
36. The composition of claim 31 , wherein the target sequence is associated with a muscle disease or disorder.
37. The composition of claim 36 , wherein the target sequence is associated with Cystic Fibrosis or Duchenne Muscular Dystrophy.
38. The composition of claim 31 , wherein the target sequence is associated with a cancer.
39. The composition of claim 38 , wherein the target sequence is associated with PD1, CTLA4, TRAC, TRBC, B2M, or MHC2TA.
40. The composition of claim 38 , wherein the composition comprises or encodes multiple engineered guides each engineered to hybridize with a different target sequence.
Assignments (6)
LICENSE
Recorded Mar 24, 2025
From: BROAD INSTITUTE, INC.
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 070609/0376 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Oct 30, 2020
From: ZHANG, FENG
To: THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 054226/0385 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Oct 30, 2020
From: ZETSCHE, BERND
To: THE BROAD INSTITUTE, INC.
Reel/Frame 054226/0490 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Oct 30, 2020
From: GOOTENBERG, JONATHAN S.
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 054226/0624 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Oct 30, 2020
From: ABUDAYYEH, OMAR O.
To: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 054226/0716 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Oct 30, 2020
From: SLAYMAKER, IAN
To: THE BROAD INSTITUTE, INC.
Reel/Frame 054226/0852 →
Priority Claims (1)
EP 16150428
· Jan 7, 2016
· regional
Continuity (10)
Continuation In Part
PCTUS2016038181
· Jun 17, 2016
Provisional Application
62232067
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Provisional Application
62205733
· Aug 16, 2015
Provisional Application
62201542
· Aug 5, 2015
Provisional Application
62193507
· Jul 16, 2015
Provisional Application
62181739
· Jun 18, 2015
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Exhibit 1072—Transcript of Teleconference with the Board, taken Nov. 27, 2018—
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Exhibit 1073—Errata to Transcript of Teleconference with the Board, taken Nov. 27, 2018—
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Exhibit 2004—Marraffini and Sontheimer, “CRISPR interference limits horizontal gene transfer in
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Exhibit 2005—Sinkunas et al., “In vitro reconstruction of cascade-mediated CRISPR immunity in
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Exhibit 2006—Jackson, et al., “Crystal structure of the CRISPR RNA-guided surveillance complex from
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Exhibit 2009—Cong et al., “Multiple genome engineering using CRISPR/Cas system,” Science, 339(6121): 819-823 (2013) (Author Manuscript—supplementary material available on-line)—
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Exhibit 2010—Mali et al., “Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat Biotechnol, 31(9): 833-838, (2013) (Author Manuscript—supplementar…
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Exhibit 2011—Pennisi, “The CRISPR craze,” Science, 15-17, (2013)—
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Exhibit 2012—Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression,” Cell 152, 1173-1183, (2013) (Supplementary material available on-line)—
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Exhibit 2014—Wu et al., “Target specificity of the CRISPR-Cas9 system,” Quant Biol, 2(2): 59-70, (2014) (Author Manuscript—supplementary material available on-line)—
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Exhibit 2017—Chen et al., “Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9,” Cell Stem Cell 17, 233-244 (2015) (Supplementary material available on-line)—
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Exhibit 2018—Dow et al., “Inducible in vivo genome editing with CRISPR/Cas9,” Nat Biotechnol, 33(4): 390-394, (2015) (Author Manuscript—supplementary material available on-line)—
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Exhibit 2022—Maggio et al., “Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells,” Scientific Reports, 4: 5105, 1-11, (2014) (Supplement…
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Exhibit 2023—Kwon et al., “Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC,” NComms 15315, 1-8 (2017) (Supplementary material available on-line)—
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Exhibit 2024—U.S. Patent Application Publication No. 20180148735 (published May 31, 2018) (Benson Hill Biosystems, Inc., applicant)—
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Expert Declaration of Dmitrij Frishman with Exhibits dated Aug. 12, 2019, 25 pages.
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