Title:
ENGINEERED CAS12I NUCLEASE, EFFECTOR PROTEIN AND USE THEREOF
Document Type and Number:
WIPO Patent Application WO/2022/247873
Kind Code:
A1
Abstract:
Provided is an engineered Cas12i nuclease, which comprises one or more of the following mutations based on a reference Cas12i nuclease: (1) replacing one or more amino acids interacting with PAM in the reference Cas12i nuclease with positively charged amino acids; and/or (2) replacing one or more amino acids, involved in opening the double strands of DNA, in the reference Cas12i nuclease with amino acids with aromatic rings; and/or (3) replacing one or more amino acids, which interact with a single-stranded DNA substrate and are located in an RuvC domain in the reference Cas12i nuclease, with positively charged amino acids; and/or (4) replacing one or more amino acids interacting with a DNA-RNA double helix in the reference Cas12i nuclease with positively charged amino acids; and/or (5) replacing one or more polar or positively charged amino acids interacting with the DNA-RNA double helix in the reference Cas12i nuclease with hydrophobic amino acids. The engineered Cas12i nuclease has significantly improved gene editing efficiency and/or significantly reduced off-target phenomenon compared with the reference Cas12i nuclease.
Inventors:
LI WEI (CN)
ZHOU QI (CN)
CHEN YANGCAN (CN)
HU YANPING (CN)
WANG XINGE (CN)
LUO SHENGQIU (CN)
CHEN YI (CN)
ZHOU QI (CN)
CHEN YANGCAN (CN)
HU YANPING (CN)
WANG XINGE (CN)
LUO SHENGQIU (CN)
CHEN YI (CN)
Application Number:
PCT/CN2022/095072
Publication Date:
December 01, 2022
Filing Date:
May 25, 2022
Export Citation:
Assignee:
INST ZOOLOGY CAS (CN)
BEIJING INST FOR STEM CELL AND REGENERATIVE MEDICINE (CN)
BEIJING INST FOR STEM CELL AND REGENERATIVE MEDICINE (CN)
International Classes:
C12N9/22; C12N15/113; C12Q1/68
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| WO2019201331A1 | 2019-10-24 | |||
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TSAI, SQ ET AL.: "GUIDE-seq enables genome-wide profiling of offtarget cleavage by CRISPR-Cas nucleases", NAT BIOTECHNOL., vol. 33, no. 2, 2015, pages 187 - 197
ZHANG, HENG ET AL.: "Mechanisms for Target Recognition and Cleavage by the Cas12i RNA-guided Endonuclease", NATURE STRUCTURAL & MOLECULAR BIOLOGY, vol. 27, no. 11, 7 September 2020 (2020-09-07), XP037295140, DOI: 10.1038/s41594-020-0499-0
VAN DER OOST, SCIENCE, vol. 358, no. 6366, 24 November 2017 (2017-11-24), pages 1019 - 1027
CHARPENTIERDOUDNA, NATURE, vol. 495, 2013, pages 50 - 51
"Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes", 1993, ELSEVIER, article "Overview of principles of hybridization and the strategy of nucleic acid probe assay"
NEEDLEMAN ET AL., J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
E. MEYERSW. MILLER, COMPUT. APPL BIOSCI., vol. 4, 1988, pages 11 - 17
NEEDLEMANWUNSCH, J MOL BIOL., vol. 48, 1970, pages 444 - 453
HUANG X ET AL., NATURE COMMUNICATIONS, vol. 11, no. 5241, 2020
ZHANG H ET AL., NATURE STRUCTURAL & MOLECULAR BIOLOGY, vol. 27, 2020, pages 1069 - 1076
SENTMANAT MF ET AL.: "A survey of validation strategies for CRISPR-Cas9 editing", SCIENTIFIC REPORTS, vol. 8, no. 888, 2018
WRIGHT, ADDISON V ET AL.: "Rational design of a split-Cas9 enzyme complex", PROC. NAT'L. ACAD. SCI., vol. 112, no. 10, 2015, pages 2984 - 2989, XP055283739, DOI: 10.1073/pnas.1501698112
PAULMURUGANPAULMURUGANGAMBHIR, CANCER RES, vol. 65, 15 August 2005 (2005-08-15), pages 7413
CRABTREE ET AL., CHEMISTRY & BIOLOGY, vol. 13, January 2006 (2006-01-01), pages 99 - 107
GAUDELLI ET AL., NATURE, vol. 551, 2017, pages 464 - 471
RICHTER ET AL., NATURE BIOTECHNOLOGY, vol. 38, 2020, pages 883 - 891
ZHENG ET AL., NUCLEIC ACIDS RES., vol. 45, no. 6, 2017, pages 3369 - 3377
KIM ET AL., BIOCHEMISTRY, vol. 45, 2006, pages 6407 - 6416
WOLF ET AL., EMBO J., vol. 21, 2002, pages 3841 - 3851
GRUNEBAUM ET AL., CURR. OPIN. ALLERGY CLIN. IMMUNOL., vol. 13, 2013, pages 630 - 638
FUKUI ET AL., J. NUCLEIC ACIDS, 2010, pages 260512
KOMORE ET AL., NATURE, vol. 533, no. 7603, 19 May 2016 (2016-05-19), pages 420 - 4
GAUDELLI ET AL., NATURE, vol. 551, no. 7681, 23 November 2017 (2017-11-23), pages 464 - 471
KIM ET AL., NATURE BIOTECHNOLOGY, vol. 35, no. 4, 2017, pages 371 - 377
A. ANZALONE ET AL., NATURE, vol. 576, no. 7785, 2019, pages 149 - 157
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY
APONTE-UBILLUS ET AL., APPL. MICROBIOL. BIOTECHNOL, vol. 102, no. 3, 2018, pages 1045 - 54
ZHONG ET AL., J. GENET. SYNDR. GENE THER, 2012
WEST ET AL., VIROLOGY, vol. 160, 1987, pages 38 - 47
TRATSCHIN ET AL., MOL. CELL. BIOL., vol. 5, 1985, pages 3251 - 60
UI-TEI ET AL., FEBS LETTERS, vol. 479, 2000, pages 79 - 82
BROPHYJENNIFER ANCHRISTOPHER A. VOIGT.: "Principles of genetic circuit design.", NATURE METHODS, vol. 11, no. 5, 2014, pages 508, XP055256504, DOI: 10.1038/nmeth.2926
SUNDBERGICHIKI: "Genetically Engineered Mice Handbook", 2006, CRC PRESS
GIBSON: "A Primer of Genome Science", 2004, SUNDERLAND, MASS.: SINAUER
TSAI, SQ ET AL.: "GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases", NAT BIOTECHNOL., vol. 33, no. 2, 2015, pages 187 - 197, XP055555627, DOI: 10.1038/nbt.3117
TSAI, SQ ET AL.: "GUIDE-seq enables genome-wide profiling of offtarget cleavage by CRISPR-Cas nucleases", NAT BIOTECHNOL., vol. 33, no. 2, 2015, pages 187 - 197
Attorney, Agent or Firm:
BEIJING CAI HE LAW FIRM (CN)
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