IP Library › Granted Patent US 12,264,359
Granted Patent B2
US 12,264,359 · App. 16/909,101 · Granted Apr 1, 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
C12Q1/6832C12N9/22C12N15/11C12N15/113C12Q1/6816C12N2310/20C12N2800/80
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,264,359
App. No.
16/909,101
Granted
Apr 1, 2025
Kind
B2
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 (50)

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 or 5-nt overhang.

43. The method of claim 40 , further comprising delivering a single-stranded oligodeoxynucleotide to the mammalian cell as a template for homology-directed repair of the double-stranded break.

44. The method of claim 40 , further comprising delivering a double-stranded oligodeoxynucleotide to the mammalian cell for insertion into the double-stranded break.

45. The method of claim 1 , wherein the CRISPR complex does not comprise a tracr sequence.

46. The method of claim 1 , wherein the Cpf1 guide comprises one or more RNAs comprising a stem left region having the sequence of UCUAC, CCUAC, UCCAC, or UCUGC, and a stem right region having the sequence of GUAGA, GUAGG, GUGGA, or GCAGA, wherein the stem left region is complementary to the stem right region.

47. The method of claim 46 , wherein the Cpf1 guide further comprises AAUU or AUU upstream of the stem left region, and an Uracil followed by a guide sequence downstream of the stem right region.

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 070609/0380 →
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 16048038 · Jul 27, 2018
Continuation 15844608 · Dec 17, 2017
Continuation In Part PCTUS2016038181 · Jun 17, 2016
Continuation In Part 14975085 · Dec 18, 2015
Provisional Application 62232067 · Sep 24, 2015
Provisional Application 62205733 · Aug 16, 2015
Provisional Application 62201542 · Aug 5, 2015
Provisional Application 62193507 · Jul 16, 2015
Provisional Application 62181739 · Jun 18, 2015
Related Publication 20200318173A1 · Oct 8, 2020
References Cited (293)
US 8361725B2 · Russell et al. · 2013 [cited by applicant]
US 8697359B1 · Zhang · 2014 [cited by applicant]
US 8889356B2 · Zhang · 2014 [cited by applicant]
US 9790490B2 · Zhang et al. · 2017 [cited by applicant]
US 10253365B1 · Doudna et al. · 2019 [cited by applicant]
US 10648020B2 · Zhang · 2020 [cited by examiner]
US 10669540B2 · Zhang · 2020 [cited by examiner]
US 11091798B2 · Zhang · 2021 [cited by examiner]
US 11286478B2 · Zhang et al. · 2022 [cited by applicant]
US 11634755B2 · Zhang · 2023 [cited by examiner]
US 11773432B2 · Zhang · 2023 [cited by examiner]
US 11781172B2 · Zhang · 2023 [cited by examiner]
US 12091709B2 · Zhang · 2024 [cited by examiner]
US 12110490B2 · Zhang · 2024 [cited by examiner]
US 20060035909A1 · Fuksova et al. · 2006 [cited by applicant]
US 20090291131A1 · Maclachlan et al. · 2009 [cited by applicant]
US 20140242699A1 · Zhang · 2014 [cited by applicant]
US 20140356956A1 · Church et al. · 2014 [cited by applicant]
US 20150079681A1 · Zhang · 2015 [cited by applicant]
US 20160208243A1 · Zhang et al. · 2016 [cited by applicant]
US 20170233756A1 · Begemann et al. · 2017 [cited by applicant]
US 20180282713A1 · Van Der Oost · 2018 [cited by applicant]
US 20190083656A1 · Khalili · 2019 [cited by applicant]
US 20190218602A1 · Zhang et al. · 2019 [cited by applicant]
US 20190233814A1 · Zhang et al. · 2019 [cited by applicant]
US 20190256900A1 · Zhang et al. · 2019 [cited by applicant]
US 20190264186A1 · Yamano et al. · 2019 [cited by applicant]
US 20200080112A1 · Zhang et al. · 2020 [cited by applicant]
US 20200181623A1 · Zhang · 2020 [cited by applicant]
US 20200263190A1 · Zhang et al. · 2020 [cited by applicant]
US 20200283755A1 · Zhang et al. · 2020 [cited by applicant]
US 20200318172A1 · Zhang et al. · 2020 [cited by applicant]
US 20200318173A1 · Zhang et al. · 2020 [cited by applicant]
US 20210040546A1 · Zhang et al. · 2021 [cited by applicant]
US 20210076366A1 · Jang et al. · 2021 [cited by applicant]
US 20210155911A1 · Zhang et al. · 2021 [cited by applicant]
US 20220162584A1 · Zhang et al. · 2022 [cited by applicant]
US 20220195503A1 · Zhang et al. · 2022 [cited by applicant]
CN 103261213A · 2013 [cited by applicant]
CN 104017821A · 2014 [cited by applicant]
CN 105602993A · 2016 [cited by applicant]
CN 106536729A · 2017 [cited by applicant]
EP 3009511A2 · 2016 [cited by applicant]
GB 15065097 · 2016 [cited by applicant]
RU 2501850C2 · 2009 [cited by applicant]
TW 201425580A · 2014 [cited by applicant]
WO WO2014065596A1 · 2014 [cited by applicant]
WO WO2014093622A2 · 2014 [cited by applicant]
WO WO2014093712A1 · 2014 [cited by applicant]
WO WO2014118272A1 · 2014 [cited by applicant]
WO WO2014191521A2 · 2014 [cited by applicant]
WO WO2014197568A2 · 2014 [cited by applicant]
WO WO2014197748A2 · 2014 [cited by applicant]
WO WO2014204723A1 · 2014 [cited by applicant]
WO WO2014204724A1 · 2014 [cited by applicant]
WO WO2015035139A2 · 2015 [cited by applicant]
WO WO2015089364A1 · 2015 [cited by applicant]
WO WO2015089419A2 · 2015 [cited by applicant]
WO WO2015089473A1 · 2015 [cited by applicant]
WO WO2015089486A2 · 2015 [cited by applicant]
WO WO2016049258A2 · 2016 [cited by applicant]
WO WO2016094872A1 · 2016 [cited by applicant]
WO WO2016166340A1 · 2016 [cited by applicant]
WO WO2016201155A1 · 2016 [cited by applicant]
WO WO2016205711A1 · 2016 [cited by applicant]
WO WO2016205749A1 · 2016 [cited by applicant]
WO WO2016205764A1 · 2016 [cited by applicant]
WO WO2017064546A1 · 2017 [cited by applicant]
WO WO2017106657A1 · 2017 [cited by applicant]
WO WO2017127807A1 · 2017 [cited by applicant]
Addgene: pY010 (pcDNA3.1-hAsCpf1) was a gift from Feng Zhang (Addgene plasmid# 69982; http://n2t.neUaddgene:69982; RRID: Addgene_69982), available online Oct. 17, 2015 (printed as pp. 1/3-3/3). [cited by applicant]
Suplatov et al., “Robust enzyme design: Bioinformatic tools for improved protein stability,” Biotechnology Journal, 2015 vol. 10 (pp. 344-355). [cited by applicant]
3rd Party Citation D46 from EP Opposition Proceedings for EP Pat. No. 3009511—Barrangou, “Class 2 large effector protein architectures”, PowerPoint Presentation Slide, Jun. 20, 2015, 1 page. [cited by applicant]
3rd Party Citation D47 from EP Opposition Proceedings for EP Pat. No. 3009511—CRIPSR Conference 2015, Website Print-out of Event Announcement, New York, New York, Jun. 18-20, 2015, accessed at https://web.archive.org/we… [cited by applicant]
3rd Party Citation D48 from EP Opposition Proceedings for EP Pat. No. 3009511—CRIPSR Conference 2015, Conference Program including Schedule of Events, New York, New York, Jun. 18-20, 2015, accessible at https://web.arch… [cited by applicant]
3rd Party Citation D49 from EP Opposition Proceedings for EP Pat. No. 3009511—Barrangou, et al., “Class 2 large effector protein architectures”, Caribou Biosciences Mail—New Systems with PowerPoint Presentation Slide, J… [cited by applicant]
3rd Party Citation D50 from EP Opposition Proceedings for EP Pat. No. 3009511—Declaration of Prof. Dr. Rodolophe Barrangou with Appendices, Aug. 13, 2019, 33 pages. [cited by applicant]
3rd Party Citation D51 from EP Opposition Proceedings for EP Pat. No. 3009511—Declaration of Andrew P. May DPhil with Appendices, Aug. 9, 2019, 24 pages. [cited by applicant]
3rd Party Citation D52 from EP Opposition Proceedings for EP Pat. No. 3009511—Declaration of Rachel E. Haurwitz, PH.D. with Appendices, Aug. 13, 2019, 8 pages. [cited by applicant]
Amit et al., “Unbiased Reconstruction of a Mammalian Transcriptional Network Mediating Pathogen Responses”, Science vol. 326, 2009, pp. 257-263. [cited by applicant]
Andreakos et al., “Distinct Pathways of LPS-INduced NF-κB Activation and Cytokine Production in Human Myeloid and Nonmyeloid Cells Defined by Selective Utilization of MyD88 and Mal/TIRAP”, Blood, vol. 103, No. 6, 2004, … [cited by applicant]
Assignment from Bernd Zetsche to The Broad Institute Inc. for U.S. Appl. No. 14/975,085 dated Jan. 5, 2016, 2 pages. [cited by applicant]
Assignment from Feng Zhang to The Broad Institute Inc. for U.S. Appl. No. 14/975,085 dated Dec. 23, 2015, 2 pages. [cited by applicant]
Assignment from Ian Slaymaker to The Broad Institute Inc. for U.S. Appl. No. 14/975,085 dated Jan. 6, 2016, 2 pages. [cited by applicant]
Assignments from Inventors, lan Slaymaker, Omar O. Abudayyeh, Feng Zhang, Jonathan Gootenberg, and Bernd Zetsche to The Broad Institute Inc. or Presidents and Fellows of Harvard College for U.S. Appl. No. 14/975,085 (an… [cited by applicant]
Charpentier et al., “Rewriting a genome”, Nature, Mar. 2013, vol. 495, 2 pages. [cited by applicant]
Chen et al., “Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting”, Nature Communications, vol. 8, Apr. 7, 2017, DOI: 10.1038/ncomms14958, pp. 1-12. [cited by applicant]
Cho, Seung Woo, et al., “Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease,” Nature Biotechnology, vol. 31 pp. 230-232, including Supplementary Information, 14 pages, 2013. [cited by applicant]
Dahlman et al., “Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease”, Nature Biotechnology, vol. 33, No. 11, Oct. 5, 2015, pp. 1159-1161, XP055381172, US. [cited by applicant]
Daligault et al., GenBank Accession No. AJJ47668, Feb. 9, 2015, 2 pages. [cited by applicant]
D'Astolfo et al., “Efficient Intracellular Delivery of Native Proteins”, Cell, vol. 161, Apr. 23, 2015, http://dx.doi.org/10.1016/j.cell.2015.03.028, pp. 674-690. [cited by applicant]
Database UniProt [Online] “SubName: Full=CRISPR-associated protein Cpf1, subtype Prefran {ECO:0000313 | EMBL: AJJ47668.1};”, XP002760659, retrieved from EBI accession No. Uniprot: A0A0B6KQP9, Database accession No. A0A0… [cited by applicant]
Database UniProt [Online] Nov. 30, 2010, SubName: Full =Uncharacterized protein {EC0:0000313:EMBL:EFL46285.1}; XP002769441, retrieved from EBI accession No. Uniprot:E1KQG5, Database accession No. E1KQG5. [cited by applicant]
Declaration of Steven R. Trybus in EPO opposition proceedings concerning European Patent No. 3 009 511 dated Sep. 14, 2018, 13 pages. [cited by applicant]
Dickey et al., “Moraxella bovoculi hypothetical protein”, Database ENA [Online] EMBL-EBI, Dec. 16, 2015, Database accession No. AKG14689, XP002772302, 3 pages. [cited by applicant]
Dickey et al., “Moraxella bovoculi hypothetical protein”, Database ENA [Online] EMBL-EBI, May 5, 2015, Database accession No. AKG12737, XP002772309, 2 pages. [cited by applicant]
Dong, et al., “The crystal structure of Cpf1 in complex with CRISPR RNA,” Nature, vol. 532, 2016, pp. 523-525. [cited by applicant]
Exhibit 1001—U.S. Pat. No. 9,790,490— [cited by applicant]
Exhibit 1002—Prosecution History of the '490 patent— [cited by applicant]
Exhibit 1003—Declaration of Dr. Chase L. Beisel and accompanying Appendices A-C— [cited by applicant]
Exhibit 1004—Schunder et al., “First indication for a functional CRISPR/Cas system in Francisella tularensis,” International Journal of Medical Microbiology, 303:51-60 (2013)— [cited by applicant]
Exhibit 1005—Zetsche et al., “Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System,” Cell, 163:759-71 (2015)— [cited by applicant]
Exhibit 1006—Zetsche et al., “A Survey of Genome Editing Activity for 16 Cpf1 orthologs,” bioRxiv, doi: https://doi.org/10.1101/134015 (2017)— [cited by applicant]
Exhibit 1007—Hsu et al., “Development and Applications of CRISPR-Cas9 for Genome Engineering,” Cell, 157:1262-78 (2014)— [cited by applicant]
Exhibit 1008—Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems,” Molecular Cell, 60:385-97 (2015)— [cited by applicant]
Exhibit 1009—Koonin et al., “Diversity, classification and evolution of CRISPR-Cas systems,” Current Opinion in Microbiology, 37:67-78 (2017)— [cited by applicant]
Exhibit 1010—Karvelis et al., “Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements,” Genome Biology, 16:253, 1-13 (2015)— [cited by applicant]
Exhibit 1011—Lowder et al., “Rapid Evolution of Manifold CRISPR Systems for Plant Genome Editing,” Frontiers in Plant Science, 7(1683):1-12 (2016)— [cited by applicant]
Exhibit 1012—Leenay et al., “Identifying and visualizing functional PAM diversity across CRISPR-Cas systems,” Mol Cell, 62(1):137-47 (2016)— [cited by applicant]
Exhibit 1013—Makarova & Koonin, “Annotation and Classification of CRISPRCas Systems,” Chapter 4 in CRISPR: Methods and Protocols, Methods in Molecular Biology, 1311:47-75 (2015)— [cited by applicant]
Exhibit 1014—HMM Summary Page: TIGR04330 (http://tigrfams.jcvi.org/cgibin/HmmReportPage.cgi?acc=TIGR04330) last visited Jun. 27, 2018— [cited by applicant]
Exhibit 1015—Begemann et al., “Characterization and Validation of a Novel Group of Type V, Class 2 Nucleases for in vivo Genome Editing,” bioRxiv, doi: http://dx.doi.org/10.1101/192799 (2017)— [cited by applicant]
Exhibit 1016—Ran et al., “In vivo genome editing using [cited by applicant]
Exhibit 1017—Kleinstiver et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities,” Nature, 523(7561):481-85 (2015)— [cited by applicant]
Exhibit 1018—Gao et al., “Engineered Cpf1 variants with altered PAM specificities increase genome targeting range,” Nature Biotechnology, 35(8):789-92 (2017)— [cited by applicant]
Exhibit 1019—Stella et al., “Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage,” Nature, 546(7659):559-63 (2017)— [cited by applicant]
Exhibit 1020—Hirano et al., “Structure and Engineering of Francisella novicida Cas9,” Cell, 164(5):950-61 (2016)— [cited by applicant]
Exhibit 1021—Fieck et al., “Modifications of the [cited by applicant]
Exhibit 1022—U.S. Pat. No. 8,697,359— [cited by applicant]
Exhibit 1023—Chiu et al., “Engineered GFP as a vital reporter in plants,” Current Biology, 6(3):325-30 (1996)— [cited by applicant]
Exhibit 1024—Mali et al., “RNA-Guided Human Genome Engineering via Cas9,” Science, 339(6121):823-26 (2013)— [cited by applicant]
Exhibit 1025—Sandy et al., “Mammalian RNAi: a practical guide,” BioTechniques, 39:215-24 (2005)— [cited by applicant]
Exhibit 1026—United States Patent Application Publication No. 2013/0302401— [cited by applicant]
Exhibit 1027—International Publication No. WO 2014/118272— [cited by applicant]
Exhibit 1028—Nair et al., “Multivalent N-Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing,” JACS, 136:16958-63 (2014)— [cited by applicant]
Exhibit 1029—Ludlum et al., “Alkylation of Synthetic Polynucleotides,” Science, 145(3630):397-99 (1964)— [cited by applicant]
Exhibit 1030—Glen Research, The Glen Report, 19(1):1-16 (2007)— [cited by applicant]
Exhibit 1031—El-Andaloussi et al., “Exosome-mediated delivery of siRNA in vitro and in vivo,” Nat Protoc, 7(12):2112-26 (2012)— [cited by applicant]
Exhibit 1032—Choulika et al., “Transfer of single gene-containing long terminal repeats into the genome of mammalian cells by a retroviral vector carrying the cre gene and the loxP site,” J Virol., 70(3):1792-98 (1996)— [cited by applicant]
Exhibit 1033—Bergemann et al., “Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination,” Nucleic Acids Research, 23(21):4451-56 (1995)— [cited by applicant]
Exhibit 1034—Dahlman et al., “In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight,” Nat Nanotechnol., 9(8):648-55 (2014)— [cited by applicant]
Exhibit 1035—Senis et al., “CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox,” Biotechnol J., 9(11):1402-12 (2014)— [cited by applicant]
Exhibit 1036—Shukla et al., “Precise genome modification in the crop species Zea mays using zinc-finger nucleases,” Nature, 459(7245):437-41 (2009)— [cited by applicant]
Exhibit 1037—Jinek et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science, 337(6069):816-21 (2012)— [cited by applicant]
Exhibit 1038—Mojica et al., “Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria,” Mol Microbiol, 36(1):244-46 (2000)— [cited by applicant]
Exhibit 1039—Ishino et al., “Nucleotide Sequence of the iap Gene, Responsible for Alkaline Phosphatase Isozyme Conversion in Escherichia coli, and Identification of the Gene Product,” Journal of Bacteriology, 169(12):54… [cited by applicant]
Exhibit 1040—Jansen et al., “Identification of genes that are associated with DNA repeats in prokaryotes,” Molecular Microbiology, 43(6):1565-75 (2002)— [cited by applicant]
Exhibit 1041—Bolotin et al., “Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin,” Microbiology, 151(Pt 8):2551-61 (2005)— [cited by applicant]
Exhibit 1042—Mojica et al., “Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements,” J Mol Evol, 60(2):174-82 (2005— [cited by applicant]
Exhibit 1043—Pourcel, “CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies,” Microbiology, 151(Pt 3): 653-3 (2005)— [cited by applicant]
Exhibit 1044—Barrangou et al., “CRISPR provides acquired resistance against viruses in prokaryotes,” Science, 315(5819):1709-12 (2007)— [cited by applicant]
Exhibit 1045—Haft et al., “A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes,” PLOS Computational Biology, 1(6):474-83 (2005)— [cited by applicant]
Exhibit 1046—Brouns et al., “Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes,” Science, 321(5891):960-64 (2008)— [cited by applicant]
Exhibit 1047—Garneau et al., “The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA,” Nature, 468(7320):67-71 (2010)— [cited by applicant]
Exhibit 1048—Deveau et al., “Phage Response to CRISPR-Encoded Resistance in [cited by applicant]
Exhibit 1049—Mojica et al., “Short motif sequences determine the targets of the prokaryotic CRISPR defence system,” Microbiology, 155(Pt3):733-40 (2009)— [cited by applicant]
Exhibit 1050—Anders et al., “Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease,” Nature, 215(7219):569-73 (2014)— [cited by applicant]
Exhibit 1051—Nishimasu et al., “Crystal Structure of Cas9 in Complex with Guide RNA and Target RNA,” Cell, 156(5):935-49 (2014)— [cited by applicant]
Exhibit 1052—Deltcheva et al., “Crispr Rna maturation by trans-encoded small RNA and host factor RNase III,” Nature, 471(7341):602-07 (2011)— [cited by applicant]
Exhibit 1053—Makarova et al., “Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems,” Biology Direct, 6:38, pp. 1-27 (2011)— [cited by applicant]
Exhibit 1054—Nam et al., “Cas5d protein process pre-crRNA and assembles into a Cascade-like interference complex in Subtype I-C/Dvulg CRISPR-Cas system,” Structure, 20(9):1574-84 (2012)— [cited by applicant]
Exhibit 1055—Haurwitz et al., “Sequence- and structure-specific RNA processing by a CRISPR endonuclease,” Science, 329(5997):1355-58 (2010)— [cited by applicant]
Exhibit 1056—Hatoum-Aslan et al., “Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site,” PNAS, 108(52):21218-2… [cited by applicant]
Exhibit 1057—Rouillon et al., “Structure of the CRISPR Interference Complex CSM Reveals Key Similarities with Cascade,” Molecular Cell, 52:124-34 (2013)— [cited by applicant]
Exhibit 1058—Hale et al., “RNA-Guided RNA Cleavage by a CRISPR RNACas Protein,” Cell, 139(5):945-56 (2009)— [cited by applicant]
Exhibit 1059—Vestergaard et al., “CRISPR adaptive immune systems of Archaea,” RNA Biology, 11(2):156-67 (2014)— [cited by applicant]
Exhibit 1060—Voskarides & Deltas, “Screening for Mutations in Kidney-Related Genes Using SURVEYOR Nuclease for Cleavage at Heteroduplex Mismatches,” Journal of Molecular Diagnostics, 11(4):311-18 (2009)— [cited by applicant]
Exhibit 1061—Findlay et al., “A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells,” PLoS One, 11(4):e0153901 (2016)— [cited by applicant]
Exhibit 1062—Kim et al., “Genotyping with CRISPR-Cas-derived RNA-guided endonucleases,” Nat Commun, 5:3157 (2014)— [cited by applicant]
Exhibit 1063—Minton, “How can biochemical reactions within cells differ from those in test tubes?,” Journal of Cell Science, 119:2863-69 (2006)— [cited by applicant]
Exhibit 1064—Ellis, “Macromolecular crowding: obvious but underappreciated,” Trends Biochem Sci, 26(10):597-604 (2001)— [cited by applicant]
Exhibit 1065—Nishimasu et al., “Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1,” Mol Cell, 67(1):139-47 (2017)— [cited by applicant]
Exhibit 1066—Shmakov et al., “Diversity and evolution of class 2 CRISPR-Cas systems,” Nat Rev Microbiol., 15(3):169-82 (2017)— [cited by applicant]
Exhibit 1067—Aravind et al., “Holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories,” Nucleic Acids Research, 28(18):3417-32 (2000)— [cited by applicant]
Exhibit 1068—Chen et al., “Structural asymmetry in the Thermus thermophilus RuvC dimer suggests a basis for sequential strand cleavages during Holiday junction resolution,” Nucleic Acids Research, 41(1):648-59 (2013)— [cited by applicant]
Exhibit 1069—Leenay & Beisel, “Deciphering, communicating, and engineering the CRISPR PAM,” J Mol Biol., 429(2):177-91 (2017)— [cited by applicant]
Exhibit 1070—Pul et al., “Identification and characterization of [cited by applicant]
Exhibit 1071—Kim et al., “Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins,” Genome Res., 24(6):1012-9 (2014)— [cited by applicant]
Exhibit 1072—Transcript of Teleconference with the Board, taken Nov. 27, 2018— [cited by applicant]
Exhibit 1073—Errata to Transcript of Teleconference with the Board, taken Nov. 27, 2018— [cited by applicant]
Exhibit 2001—Ledford, “Five big mysteries about CRISPR's origins,” Nature, 541, 7637, (2017) (last visited Oct. 5, 2018) https://www.nature.com/news/five-big-mysteries-about-crispr-sorigins-1.21294— [cited by applicant]
Exhibit 2002—Sapranauskas et al., “The [cited by applicant]
Exhibit 2003—Gasiunas et al., “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria,” PNAS, 10.1073, (2012)— [cited by applicant]
Exhibit 2004—Marraffini and Sontheimer, “CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA,” Science, 322(5909):1843-1845, (2008) (Author Manuscript—supplementary material available o… [cited by applicant]
Exhibit 2005—Sinkunas et al., “In vitro reconstruction of cascade-mediated CRISPR immunity in [cited by applicant]
Exhibit 2006—Jackson, et al., “Crystal structure of the CRISPR RNA-guided surveillance complex from [cited by applicant]
Exhibit 2007—Mulepati et al., “Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target,” Science, 345(6203):1479-1484, (2014) (Author Manuscript—supplementary material available on-line)— [cited by applicant]
Exhibit 2008—Thabet et al., “Evolutionary trends of the transposase-encoding open reading frames A and B (orfA and orfB) of the mycobacterial IS6110 Insertion sequence,” PLoS One, 10(6):1-5, (2015) (Supplementary materi… [cited by applicant]
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)— [cited by applicant]
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—supplementary… [cited by applicant]
Exhibit 2011—Pennisi, “The CRISPR craze,” Science, 15-17, (2013)— [cited by applicant]
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)— [cited by applicant]
Exhibit 2013—Gilbert et al., “CRISPR-Mediated modular RNA-guided regulation of transcription in Eukaryotes,” Cell 154, 442-451, (2013) (Supplementary material available on-line)— [cited by applicant]
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)— [cited by applicant]
Exhibit 2015—Doudna et al., “The new frontier of genome engineering with CRISPR-Cas9,” Science, 346(6213), 1077-1086 (2014)— [cited by applicant]
Exhibit 2016—O'Geen et al., “How specific is CRISPR/Cas9 really?” Current Opinion in Chemical Biology, 29:72-78-, (2015)— [cited by applicant]
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)— [cited by applicant]
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)— [cited by applicant]
Exhibit 2019—Didovyk et al., “Transcriptional regulation with CRISPR-Cas9: principles, advances and applications,” Curr Opin Biotechnol, 40: 177-184, (2016) (Author Manuscript). [cited by applicant]
Exhibit 2020—Guilinger et al., “Fusion of catalytically inactive Cas9 to Fokl nuclease improves the specificity of genome modification,” Nat Biotechnol, 32(6):577-582, (2014) (Author Manuscript—supplementary material av… [cited by applicant]
Exhibit 2021—Horvath et al., “CRISPR/Cas, the immune system of bacteria and archaea,” Science 327, 167-170, (2010)— [cited by applicant]
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) (Supplementa… [cited by applicant]
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)— [cited by applicant]
Exhibit 2024—U.S. Patent Application Publication No. 20180148735 (published May 31, 2018) (Benson Hill Biosystems, Inc., applicant— [cited by applicant]
Expert Declaration of Dmitrij Frishman with Exhibits dated Aug. 12, 2019, 25 pages. [cited by applicant]
Fagerlund et al., “The Cpf1 CRISPR-Cas protein expands genome-editing tools”, Genome Biology, vol. 16, No. 251, Nov. 7, 2015, DOI 10.1186/s13059-015-0824-9, pp. 1-3. [cited by applicant]
Flatman, “Magnesium Transport across Cell Membranes”, Journal of Membrane Biology, 80, 1984, pp. 1-14. [cited by applicant]
Gao, et al. “Engineered Cpf1 variants with altered PAM specificities”, Nature Biotechnology, vol. 35, Jun. 8, 2017, pp. 1-4 (789-792), doi:10.1038/nbt.3900, advanced online publication including Supplementary Informatio… [cited by applicant]
GEO Accession No. GSE2706 (Expression Profile Analysis for Tank, obtained from http://www.ncbi.nlm.nih.gov/geo/ tools/profileGraph.cgi?ID=GDS1249:207616_s_at, Nov. 7, 2014, 2 pages. [cited by applicant]
Ghosh et al., “Toll-like Receptor (TLR) 2-9 Agonists-Induced Cytokines and Chemokines: I. Comparison with T Cell Receptor-Induced Responses”, Cellular Immunology, vol. 243, 2006, pp. 48-57. [cited by applicant]
Graham et al., “Resources for the design of CRISPR gene editing experiments”, Genome Biology, vol. 16, No. 260, Nov. 27, 2015, DOI 10.1186/s13059-015-0823-x, pp. 1-21. [cited by applicant]
Guilinger et al., “Fusion of catalytically inactive Cas9 to Fokl nuclease improves the specificity of genome modification”, Nature Biotechnology, Jun. 2014, vol. 32, No. 6, pp. 577-581. [cited by applicant]
Guo, et al., “Protein Tolerance to Random Amino Acid Change”, The National Academy of Sciences, PNAS, Jun. 22, 2004, vol. 101, No. 25, pp. 9205-9210. [cited by applicant]
Haft, D.H., “HMM Summary Page: TIGR04330”, 2012, XP-002757584, http://jcvi.org/cgi-bin/tigrfams/HmmReportPage.cgi?acc=TIGR04330, 1 page. [cited by applicant]
Hsu, et al., “DNA targeting specificity of RNA-guided Cas9 nucleases,” Nature Biotechnology, vol. 31, 2013, pp. 827-834. [cited by applicant]
Hwang W., et al., “Efficient In Vivo Genome Editing Using RNA-Guided Nucleases,” Nature Biotechnology, vol. 31, No. 3, Mar. 2013, pp. 227-229 (12 pages). [cited by applicant]
Jiang et al., “Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in [cited by applicant]
Kawagoe et al., “Tank is a Negative Regulator of Toll-Like Receptor Signaling and is Critical for the Prevention of Autoimmune Nephritis”, Nature Immunology, vol. 10, No. 9, 2009, pp. 965-973. [cited by applicant]
Kim et al., “Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells”, Nature Biotechnology, vol. 34, No. 8, Jun. 6, 2016 (corrected Jul. 18, 2016), pp. 863-888. [cited by applicant]
Kleinstiver, et al. “Engineered CRISPR-Cas9 nucleases with altered PAM specificities, ” Nature, vol. 523, vol. 523, 2015, pp. 1-27, 27 p. [cited by applicant]
Krawczyk et al., “Toll-like Receptor-Induced Changes in Glycolytic Metabolism Regulate Dendritic Cell Activation”, Blood, vol. 115, No. 23, 2010, pp. 4742-4749. [cited by applicant]
Leenay, et al., “Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems”, Molecular Cell, vol. 62, 2016, pp. 137-147, 11 p. [cited by applicant]
Liu et al., “C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism”, Molecular Cell, vol. 65, Jan. 19, 2017, pp. 310-322. [cited by applicant]
Liu et al., “Two Distant Catalytic Sites Are Responsible for C2c2 RNase Activities”, Cell, vol. 168, Jan. 12, 2017, pp. 121-134. [cited by applicant]
Lund et al., “Toll-like Receptor 9-Mediated Recognition of Herpes Simplex Virus-2 by Plasmacytoid Dendritic Cells”, J. Exp. Med., vol. 198, No. 3, Aug. 4, 2003, pp. 513-520. [cited by applicant]
Lundgren et al., Methods in Molecular Biology, vol. 1311, Chapters 1 and 4, 2015, pp. 1-21 and 47-75. [cited by applicant]
Makarova, et al., “An updated evolutionary classification of CRISPR-Cas systems” Nature Reviews-Microbiology, vol. 13 2015, pp. 722-736, 15 p. [cited by applicant]
Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering”, Nature Biotechnology, vol. 31, 2013, pp. 833-837, 5 pages. [cited by applicant]
Mastroianni, et al., “Group II Intron-Based Gene Targeting Reactions in Eukaryotes” Plos One, vol. 3, 2008, pp. 1-15, 15 pages. Doi:10.1371/journal.pone.0003121. [cited by applicant]
Naito et al., “CRISPRdirect: Software for designing CRISPR/Cas guide RNA with reduced off-target sites”, Bioinformatics, 2015, vol. 31, No. 7, pp. 1120-1123. [cited by applicant]
Napolitani et al., “Selected Toll-like Receptor Agonist Combinations Synergistically Trigger a T Helper Type 1-Polarizing Program in Dendritic Cells”, Nature Immunology, vol. 6, No. 8, 2005, pp. 769-776. [cited by applicant]
New England Biolabs, “NEBuffer 3 product information”, Feb. 26, 2018, 3 pages. [cited by applicant]
Nishimasu et al., “Crystal Structure of Cas9 in Complex with Guide RNA and Target RNA,” Cell, 156(5):935-49 (2014). [cited by applicant]
Nishimasu et al., “Crystal Structure of [cited by applicant]
Pattanayak et al., “High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity”, Nature Biotechnology, Sep. 2013, vol. 31, No. 9, pp. 839-845. [cited by applicant]
Prakash et al., “Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice”, Nucleic Acids Research, vol. 42, No. 13, Jul. 3, 2014, doi: 10… [cited by applicant]
Ran et al., “Double Nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity”, Cell, Sep. 12, 2013, vol. 154, pp. 1380-1389. [cited by applicant]
Reindl et al., “Abstract: A Pan-Specific Inhibitor of the Polo-Box Domains of Polo-Like Kinases Arrests Cancer Cells in Mitosis”, Chem Bio Chem, vol. 10, Apr. 6, 2009, pp. 1145-1148. Downloaded from http://onlinelibrary… [cited by applicant]
Ruse et al., “New Players in TLR-Mediated Innate Immunity: P13K and Small Rho GTPases”, Immunologic Research, vol. 34, 2006, pp. 33-48. [cited by applicant]
Sadowski et al., “The sequence-structure relationship and protein function prediction”, Current Opinion in Structural Biology, vol. 19 pp. 2009, 357-362 (6 pages). [cited by applicant]
Sander, et al., “CRISPR-Cas systems for editing, regulating and targeting genomes,” Nature Biotechnology, vol. 32, 2014, pp. 347-355, 9 pages. [cited by applicant]
Schunder et al., “First indication for a functional CRISPR/Cas system in Francisella tularensis”, International Journal of Medical Microbiology, vol. 303, 2013, pp. 51-60, 10 pages. [cited by applicant]
ScienceDirect Excerpt—D'Astolfo et al., “Efficient Intracellular Delivery of Native Proteins”, Cell, vol. 161, Issue 3, Apr. 23, 2015, pp. 674-690 (article provided separately—excerpt 1 page only). [cited by applicant]
Seffernick et al., “Melamine Deaminase and Atrazine Chlorohydrolase: 98 Percent Identical but Functionally Different”, Journal Bacteriology, vol. 183, 2001, 2405-2410, (6 pages). [cited by applicant]
Sequence listing filed for EP Application No. 16150428.7 on Jan. 7, 2016, pp. 1-1286. [cited by applicant]
Shmakov et al, “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell, vol. 60, No. 3, Oct. 22, 2015. pages 385-397, XP055267512, US. [cited by applicant]
Slaymaker et al., “Rationally engineered Cas9 nucleases with improved specificity”, Science, vol. 351, No. 6268, Jan. 1, 2016, pp. 84-88. [cited by applicant]
Strebhardt, “Multifaceted Polo-like Kinases: Drug Targets and Antitargets for Cancer Therapy”, Nature Reviews, vol. 9, Aug. 2010, pp. 643-660. [cited by applicant]
Supporting Online Material (43 pages) for Amit et al., “Unbiased Reconstruction of a Mammalian Transcriptional Network Mediating Pathogen Responses”, Science, vol. 326, 2009, pp. 257-263. [cited by applicant]
Takashi et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA”, Cell, Apr. 2016, vol. 165, No. 4, pp. 949-962. [cited by applicant]
Tang et al., “Identification of Dehalobacter reductive dehalogenases that catalyse dichlorination of chloroform, 1,1,1-trichloroethane and 1,2-dichloroethane”, Philosophical Transactions of the Royal Society B, vol. 368… [cited by applicant]
Termeer et al., “Oligosaccharides of Hyaluronan Activate Dendritic Cells via Toll-like Receptor 4”, Journal of Experimental Medicine, vol. 195, No. 1, 2002, pp. 99-111. [cited by applicant]
Type V CRISPR-associated protein Cpfi [ [cited by applicant]
Van Der Oost et al., “Unravelling the structural and mechanistic basis of CRISPR-Cas systems”, Nature Reviews, Microbiology, Jul. 2014, vol. 12, pp. 479-492. [cited by applicant]
Van Der Oost, “New tool for genome surgery”, Science, vol. 339, Feb. 15, 2013, pp. 768-770, 3 pages. [cited by applicant]
Vestergaard et al.:. , “CRISPR adaptive immune systems of Archaea”, RNA Biology, vol. 11,2014, pp. 156-167, 12 pages. [cited by applicant]
Wiedenfeft et al., “Structural Basis for DNase Activity of a Conserved Protein Implicated in CRISPR-Mediated Genome Defense”, Structure, 17, 2009, pp. 904-912. [cited by applicant]
Witkowski et al., “Conversation of a β-Ketoacyl Synthase to a Malonyl Decarboxylase by Replacement of the Active-Site Cysteine with Glutamine”, Biochemistry, vol. 38, 1999, pp. 11643-11650. [cited by applicant]
Xie, et al. “RNA-Guided Genome Editing in Plants Using a CRISPR-Cas System” Molecular Plant, vol. 6, Nov. 2013, 1975-1983, 9 pages. [cited by applicant]
Xie, K, et al., “Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system”, Proc. Natl. Acad. Sci. U.S.A., 2015, vol. 112. pp. 3570-3575. [cited by applicant]
Yang et al., “Making and Breaking Nucleic Acids: Two-Mg2-Ion Catalysis and Substrate Specificity”, Molecular Cell, vol. 22, Apr. 7, 2006, pp. 5-13. [cited by applicant]
Yang et al., “PAM-Development Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease”, Cell, vol. 167, Dec. 15, 2016, pp. 1814-1828. [cited by applicant]
Zetsche et al., “Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system”, Cell, vol. 163, Oct. 22, 2015, pp. 759-771, 13 pages. [cited by applicant]
Pougach, K.S., et al., “CRISPR Adaptive Immunity Systems of Prokaryotes,” Molecular Biology, 2012, vol. 46 (pp. 175-182). [cited by applicant]
Rohmer et al., Gen Bank accession No. AOQ7Q2, Dec. 9, 2015 (3 pages). [cited by applicant]
Singh et al., “Protein Engineering Approaches in the Post-Genomic Era,” Current Protein and Peptide Science, 2018, vol. 19, No. 1 (pp. 5-15). [cited by applicant]
Bikard et al., “Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system,” Nucleic Acids Research, 2013, vol. 41, No. 15 (pp. 7429-7437). [cited by applicant]
Cai et al., “Targeted transgene integration in plant cells using designed zinc finger nucleases,” Plant Molecular Biology, 2009, vol. 69 (pp. 699-709). [cited by applicant]
Gao, L., et al., Engineered Cpf1 enzymes with altered PAM specificities. bioRxiv preprint doi: https://doi.org/10.1101/091611, Dec. 4, 2016, pp. 1/14, 14/14, pp. 1/3-3/3 of Figs, and pp. S1-S8. [cited by applicant]
Gen Bank Accession No. A0Q7Q2.1, RecName: Full=CRISPR-associated endonuclease Cpf1; AltName: Full=FnCpf1, publicly available Dec. 2015, printed as pp. 1/2-2/2. (Year: 2015). [cited by applicant]
Gen Bank Accession No. U2UMQ6.1, Rec Name: Full=CRISPR-associated endonuclease Cpf1; AltName: Full=AsCpf1, publicly available Dec. 2015, printed as pp. 1/2-2/2. (Year: 2015). [cited by applicant]
Gen Bank Accession No. WP _051666128.1, publicly available Aug. 16, 2015, printed as p. 1/1. [cited by applicant]
Ledfrod, “Bacteria yield new gene cutter,” Nature, Oct. 1, 2015, vol. 526 (p. 17). [cited by applicant]
Lee et al., “Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway,” Scientific Reports, Feb. 25, 2015, vol. 5 (pp. 1-11). [cited by applicant]
Leinonen et al., “The EMBL sequence version archive,” Bioinformatics, 2003 vol. 19, No. 14 (pp. 1861-1862). [cited by applicant]
Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression,” Cell, Feb. 28, 2013, vol. 152 (pp. 1173-1183). [cited by applicant]
Wang et al., “Delivery of CRISPR/Cas9 by Novel Strategies for Gene Therapy,” ChemBioChem,2019 vol. 20 (pp. 634-643). [cited by applicant]
Wu et al., “Correction of a Genetic Disease in Mouse via Use of CRISPR-Cas9,” Cell Stem Cell, Brief Report, vol. 13, Dec. 5, 2013, pp. 659-662. [cited by applicant]
Yamano et al., “Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA,” Cell, vol. 165, May 5, 2016 (pp. 949-962), including pp. S1-S7 of Supplemental Figures, and pp. 1-2 of Supplemental Information. [cited by applicant]
Zhu et al., “A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells,” Stem Cell Reports, Jun. 9, 2015, vol. 4, (pp. 1103-1111). [cited by applicant]
Calero-Garcia et al., “Gene-ectomy: Gene Ablation with CRISPR/Cas9 in Human Hematopoietic Cells,” Cell Stem Cell, Nov. 6, 2014 (2 pages). [cited by applicant]
Long et al., “Prevention of muscular dystrophy in mice by CRISPR/Cas9 mediated editing of germline DNA,” Science, Sep. 5, 2014, vol. 345, No. 6201 (pp. 1184-1188). [cited by applicant]
Doudna et al., “The new frontier of genome engineering with CRISPR-Cas9,” Science, Nov. 28, 2014, vol. 346 (pp. 1077-1087). [cited by applicant]
Konermann et al., “Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex,” Nature, Jan. 29, 2015, vol. 517, pp. 583-588 (18 pages). [cited by applicant]
Cong et al., “CRISPR-Assisted Mammalian Genome Engineering,” published as “Multiplex Genome Engineering Using CRISPR-Cas Systems,” Science, Oct. 5, 2012, vol. 339 (pp. 819-823) [Manuscript including Supplementary Materi… [cited by applicant]
Mali et al., “RNA-Guided Human Genome Engineering via Cas9” Science, dated Feb. 15, 2013, vol. 339 (pp. 823-826, 41 p.—Includes Supplemental Information). [cited by applicant]
Gen Bank Accession No. KDN25524.1, publicly available May 2014, printed as pp. 1/2-2/2. [cited by applicant]
Gen Bank Accession No. KIE18657.1, publicly available Jan. 2015, printed as pp. 1/2-2/2. [cited by applicant]
Gen Bank Accession No. KKP36646.1, publicly available May 2015, printed as pp. 1/2-2/2. [cited by applicant]
Gen Bank Accession No. KKQ36153.1, publicly available May 2015, printed as pp. 1/2-2/2. [cited by applicant]
Gen Bank Accession No. KKQ38174.1, publicly available May 2015; printed as pp. 1/3-3/3. [cited by applicant]
Gen Bank Accession No. KKR91555.1 publicly available May 2015, printed as pp. 1/2-2/2. [cited by applicant]
Gen Bank Accession No. KKT48220.1, publicly available May 2015, printed as pp. 1/2-2/2. [cited by applicant]
Gen Bank Accession No. WP 006283774.1, publicly available Nov. 2023, printed as p. 1/1. [cited by applicant]
Gen Bank Accession No. WP 009217842.1, publicly available Oct. 2019, printed as p. 1/1. [cited by applicant]
Gen Bank Accession No. WP 016301126.1, publicly available Oct. 2019, printed as p. 1/1. [cited by applicant]
Gen Bank Accession No. WP 02710509.1, publicly available Oct. 13, 2019, printed as p. 1/1. [cited by applicant]
Gen Bank Accession No. WP 044910713.1, publicly available Oct. 2019, printed as p. 1/1. [cited by applicant]
Gen Bank Accession No. WP 044919442.1, publicly available Oct. 2019, printed as p. 1/1. [cited by applicant]
Gen Bank Accession No. WP 04910712.1, publicly available Oct. 2019, printed as p. 1/1. [cited by applicant]
Gen Bank Accession No. WP 050786240.1, publicly available Oct. 2019, printed as p. 1/1. [cited by applicant]
Gen Bank Accession No. WP 051666128.1, publicly available 2019, printed as p. 1/1. [cited by applicant]
Zetsche et al., “Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system,” Cell, Oct. 22, 2015, vol. 163, No. 3 (pp. 759-771) [with Supplemental Information]—36 Pages). [cited by applicant]
Cited By (1)
US 12,686,862