IP Library › Granted Patent US 12,378,549
Granted Patent B2
US 12,378,549 · App. 17/054,008 · Granted Aug 5, 2025

CRISPR-cas9 system and uses thereof

Inventor: Yannick Doyon (Québec, CA)
Assignee: UNIVERSITÉ LAVAL
C12N15/111C12N9/22C12N2310/122C12N2310/20
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Quick Facts
Patent No.
US 12,378,549
App. No.
17/054,008
Granted
Aug 5, 2025
Kind
B2
Abstract

Methods and products are described herein for the modification of nucleic acids using a CRISPR/Cas9 system. Also described herein are uses of such methods and products for the modification of a target nucleic acid in a cell, in vitro or in vivo. Such methods and products may also be used for prevention or treatment of a condition associated with a target polynucleotide.

Claims (6)

1. An isolated hybrid clustered regularly interspaced short palindromic repeats (CRISPR) nuclease polypeptide comprising guide RNA (gRNA)-binding and nuclease domains from Streptococcus thermophilus strain LMD-9, and a protospacer adjacent motif (PAM)-interacting domain from Streptococcus thermophilus strain LMG18311, CNRZ1066 or TH1477, wherein the hybrid CRISPR nuclease polypeptide comprises the amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 40 or SEQ ID NO: 44.

2. A nucleic acid comprising a nucleotide sequence encoding the isolated polypeptide of claim 1 .

3. A vector comprising the nucleic acid of claim 2 .

4. The isolated polypeptide of claim 1 , comprising the amino acid sequence of SEQ ID NO: 36.

5. The isolated polypeptide of claim 1 , comprising the amino acid sequence of SEQ ID NO: 40.

6. The isolated polypeptide of claim 1 , comprising the amino acid sequence of SEQ ID NO: 44.

Assignments (1)
NUNC PRO TUNC ASSIGNMENT Recorded Nov 9, 2020
From: DOYON, YANNICK
To: UNIVERSITÉ LAVAL
Reel/Frame 054316/0289 →
Continuity (2)
Provisional Application 62670135 · May 11, 2018
Related Publication 20220119809A1 · Apr 21, 2022
References Cited (96)
US 20160355797A1 · Konermann et al. · 2016 [cited by applicant]
US 20170121693A1 · Liu · 2017 [cited by examiner]
EP 3705490B1 · 2024 [cited by applicant]
WO 2016089433A1 · 2016 [cited by applicant]
Nishimasu et al. Crystal Structure of [cited by examiner]
Chimeric. NCI Dictionary of Cancer Terms. National Cancer Institute (accessed at: https://web.archive.org/web/20180423080722/ https://www.cancer.gov/publications/dictionaries/cancer-terms/def/chimeric) (Year: 2018). [cited by examiner]
Derived. Dictionary.com (accessed at https://www.dictionary.com/browse/derived on Aug. 1, 2024) (Year: 2024). [cited by examiner]
Komor, A.C., Badran, A.H. & Liu, D.R. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell 169, 559 (2017). [cited by applicant]
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012). [cited by applicant]
Koonin, E.V., Makarova, K.S. & Zhang, F. Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol 37, 67-78 (2017). [cited by applicant]
Shmakov, S. et al. Diversity and evolution of class 2 CRISPR-Cas systems. Nat Rev Microbiol 15, 169-182 (2017). [cited by applicant]
Hille, F. et al. The Biology of CRISPR-Cas: Backward and Forward. Cell 172, 1239-1259 (2018). [cited by applicant]
Esvelt, K.M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods 10, 1116-1121 (2013). [cited by applicant]
Ran, F.A. et al. In vivo genome editing using [cited by applicant]
Chen, F. et al. Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting. Nat Commun 8, 14958 (2017). [cited by applicant]
Anderson, E.M. et al. Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition. PLoS One 13, e0192181 (2018). [cited by applicant]
Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759-771 (2015). [cited by applicant]
Colella, P., Ronzitti, G. & Mingozzi, F. Emerging Issues in AAV-Mediated In Vivo Gene Therapy. Mol Ther Methods Clin Dev 8, 87-104 (2018). [cited by applicant]
Friedland, A.E. et al. Characterization of [cited by applicant]
Kim, E. et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat Commun 8, 14500 (2017). [cited by applicant]
Ibraheim, R. et al. All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo. bioRxiv (2018). [cited by applicant]
Mir, A., Edraki, A., Lee, J. & Sontheimer, E.J. Type II-C CRISPR-Cas9 Biology, Mechanism, and Application. ACS Chem Biol 13, 357-365 (2018). [cited by applicant]
Anguela, X.M. et al. Robust ZFN-mediated genome editing in adult hemophilic mice. Blood 122, 3283-3287 (2013). [cited by applicant]
Li, H. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217-221 (2011). [cited by applicant]
Sharma, R. et al. In vivo genome editing of the albumin locus as a platform for protein replacement therapy. Blood 126, 1777-1784 (2015). [cited by applicant]
Nami, F. et al. Strategies for In Vivo Genome Editing in Nondividing Cells. Trends Biotechnol (2018). [cited by applicant]
He Z, Proudfoot C, Mileham A, J., McLaren DG, Whitelaw BA, Lillico SG. Highly efficient targeted chromosome deletions using CRISPR/Cas9. Biotechnology and Bioengineering. 112(5): 1060-4 (2015). [cited by applicant]
Byrne SM, Ortiz L, Mali P, Aach J, Church GM. Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Res. 43(3):e21 (2015). [cited by applicant]
Schneller, J.L., Lee, C.M., Bao, G. & Venditti, C.P. Genome editing for inborn errors of metabolism: advancing towards the clinic. BMC Med 15, 43 (2017). [cited by applicant]
Au, C.H. & Suh, Y. In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease. F1000Res 6, 2153 (2017). [cited by applicant]
Bolotin, A. et al. Complete sequence and comparative genome analysis of the dairy bacterium [cited by applicant]
Barrangou, R. & Horvath, P. A decade of discovery: CRISPR functions and applications. Nat Microbiol 2, 17092 (2017). [cited by applicant]
Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551-2561 (2005). [cited by applicant]
Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709-1712 (2007). [cited by applicant]
Horvath, P. et al. Diversity, activity, and evolution of CRISPR loci in [cited by applicant]
Deveau, H. et al. Phage response to CRISPR-encoded resistance in [cited by applicant]
Garneau, J.E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67-71 (2010). [cited by applicant]
Briner, A.E. et al. Guide RNA functional modules direct Cas9 activity and orthogonality. Mol Cell 56, 333-339 (2014). [cited by applicant]
Hynes, A.P. et al. An anti-CRISPR from a virulent [cited by applicant]
Chari, R., Mali, P., Moosburner, M. & Church, G.M. Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nat Methods 12, 823-826 (2015). [cited by applicant]
Kleinstiver, B.P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481-485 (2015). [cited by applicant]
Agudelo, D. et al. Marker-free coselection for CRISPR-driven genome editing in human cells. Nat Methods 14, 615-620 (2017). [cited by applicant]
Dalvai, M. et al. A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells. Cell Rep 13, 621-633 (2015). [cited by applicant]
Guschin, D.Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 649, 247-256 (2010). [cited by applicant]
Brinkman, E.K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42, e168 (2014). [cited by applicant]
Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17, 148 (2016). [cited by applicant]
Tomoeda, K. et al. Mutations in the 4-hydroxyphenylpyruvic acid dioxygenase gene are responsible for tyrosinemia type III and hawkinsinuria. Mol Genet Metab 71, 506-510 (2000). [cited by applicant]
Ruetschi, U. et al. Mutations in the 4-hydroxyphenylpyruvate dioxygenase gene (HPD) in patients with tyrosinemia type II. Hum Genet 106, 654-662 (2000). [cited by applicant]
Russell, S. et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated Inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet 390, 849-860 (2017… [cited by applicant]
George, L.A. et al. Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant. N Engl J Med 377, 2215-2227 (2017). [cited by applicant]
Nathwani, A.C. et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med 371, 1994-2004 (2014). [cited by applicant]
McKay, T.R. et al. Perinatal gene transfer to the liver. Curr Pharm Des 17, 2528-2541 (2011). [cited by applicant]
Wang, L. et al. AAV8-mediated hepatic gene transfer in infant rhesus monkeys ( [cited by applicant]
Wang, L., Wang, H., Bell, P., McMenamin, D. & Wilson, J.M. Hepatic gene transfer in neonatal mice by adeno-associated virus serotype 8 vector. Hum Gene Ther 23, 533-539 (2012). [cited by applicant]
Yang, Y. et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol 34, 334-338 (2016). [cited by applicant]
Morrow, G. & Tanguay, R.M. Biochemical and Clinical Aspects of Hereditary Tyrosinemia Type 1. Adv Exp Med Biol 959, 9-21 (2017). [cited by applicant]
Grompe, M. Fah Knockout Animals as Models for Therapeutic Liver Repopulation. Adv Exp Med Biol 959, 215-230 (2017). [cited by applicant]
Endo, F. et al. Complete rescue of lethal albino c14COS mice by null mutation of 4-hydroxyphenylpyruvate dioxygenase and induction of apoptosis of hepatocytes in these mice by in vivo retrieval of the tyrosine catabolic… [cited by applicant]
Pankowicz, F.P. et al. Reprogramming metabolic pathways in vivo with CRISPR/Cas9 genome editing to treat hereditary tyrosinaemia. Nat Commun 7, 12642 (2016). [cited by applicant]
Nathwani, A.C. et al. Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor X expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blo… [cited by applicant]
McIntosh, J. et al. Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant. Blood 121, 3335-3344 (2013). [cited by applicant]
Pawluk, A., Davidson, A.R. & Maxwell, K.L. Anti-CRISPR: discovery, mechanism and function. Nat Rev Microbiol 16, 12-17 (2018). [cited by applicant]
Shin, J. et al. Disabling Cas9 by an anti-CRISPR DNA mimic. Sci Adv 3, e1701620 (2017). [cited by applicant]
Rauch, B.J. et al. Inhibition of CRISPR-Cas9 with Bacteriophage Proteins. Cell 168, 150-158 e110 (2017). [cited by applicant]
Rousseau, B.A., Hou, Z., Gramelspacher, M.J. & Zhang, Y. Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis. Mol Cell 69, 906-914 e904 (2018). [cited by applicant]
Strutt, S.C., Torrez, R.M., Kaya, E., Negrete, O.A. & Doudna, J.A. RNA-dependent RNA targeting by CRISPR-Cas9. Elife 7 (2018). [cited by applicant]
Zhang, Y., Rajan, R., Seifert, H.S., Mondragon, A. & Sontheimer, E.J. DNase H Activity of Neisseria meningitidis Cas9. Mol Cell 60, 242-255 (2015). [cited by applicant]
Stephenson, A.A., Raper, A.T. & Suo, Z. Bidirectional Degradation of DNA Cleavage Products Catalyzed by CRISPR/Cas9. J Am Chem Soc 140, 3743-3750 (2018). [cited by applicant]
Ma, E., Harrington, L.B., O'Connell, M.R., Zhou, K. & Doudna, J.A. Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes. Mol Cell 60, 398-407 (2015). [cited by applicant]
Dugar, G. et al. CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9. Mol Cell 69, 893-905 e897 (2018). [cited by applicant]
Chen, J.S. et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science (2018). [cited by applicant]
Boettcher, M. et al. Dual gene activation and knockout screen reveals directional dependencies in genetic networks. Nat Biotechnol 36, 170-178 (2018). [cited by applicant]
Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res 42, 2577-2590 (2014). [cited by applicant]
Goudy, K.S., Annoni, A., Naldini, L. & Roncarolo, M.G. Manipulating Immune Tolerance with Micro-RNA Regulated Gene Therapy. Frontiers in microbiology 2, 221 (2011). [cited by applicant]
Charlesworth, C.T. et al. Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans. bioRxiv (2018). [cited by applicant]
Moreno, A.M. et al. Exploring protein orthogonality in immune space: a case study with AAV and Cas9 orthologs. bioRxiv (2018). [cited by applicant]
Wagner, D.L. et al. High prevalence of [cited by applicant]
Chew, W.L. et al. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat Methods 13, 868-874 (2016). [cited by applicant]
Muller, M. et al. [cited by applicant]
Karvelis, T., Gasiunas, G. & Siksnys, V. Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: A brief overview. Methods 121-122, 3-8 (2017). [cited by applicant]
Leenay, R.T. & Beisel, C.L. Deciphering, Communicating, and Engineering the CRISPR PAM. J Mol Biol 429, 177-191 (2017). [cited by applicant]
Rock, J.M. et al. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nat Microbiol 2, 16274 (2017). [cited by applicant]
Makarova, K. et al. Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci U S A 103, 15611-15616 (2006). [cited by applicant]
Chen, H., Choi, J. & Bailey, S. Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease. J Biol Chem 289, 13284-13294 (2014). [cited by applicant]
Hu, J.H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57-63 (2018). [cited by applicant]
Kleinstiver, B.P. et al. Broadening the targeting range of [cited by applicant]
Chatterjee, P., Jakimo, N. & Jacobson, J.M. Divergent PAM Specificity of a Highly-Similar SpCas9 Ortholog. bioRxiv (2018). [cited by applicant]
Gray, S.J. et al. Production of recombinant adeno-associated viral vectors and use in in vitro and in vivo administration Curr Protoc Neurosci Chapter 4, Unit 4 17 (2011). [cited by applicant]
Aurnhammer, C. et al. Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Methods 23, 18-28 (2012). [cited by applicant]
Grompe, M. et al. Loss of fumarylacetoacetate hydrolase is responsible for the neonatal hepatic dysfunction phenotype of lethal albino mice. Genes Dev 7, 2298-2307 (1993). [cited by applicant]
Yardeni, T., Eckhaus, M., Morris, H.D., Huizing, M. & Hoogstraten-Miller, S. Retro-orbital injections in mice. Lab Anim (NY) 40, 155-160 (2011). [cited by applicant]
Cyr, D., Giguere, R., Villain, G., Lemieux, B. & Drouin, R. A GC/MS validated method for the nanomolar range determination of succinylacetone in amniotic fluid and plasma: an analytical tool for tyrosinemia type I. J Ch… [cited by applicant]
Chatterjee, P., Jakimo, N. & Jacobson, J.M. Minimal PAM specificity of a highly similar SpCas9 ortholog. Sci Adv 4, eaau0766 (2018). [cited by applicant]
International Search Report and Written Opinion dated Aug. 9, 2019 in respect of PCT/CA2019/050629. [cited by applicant]
International Preliminary Report on Patentability dated Nov. 17, 2020 in respect of PCT/CA2019/050629. [cited by applicant]
Ma et al., Engineer chimeric Cas9 to expand PAM recognition based on evolutionary information, Nature Communications 10(1): 1-9 (2019). [cited by applicant]