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Engineering of CRISPR-Cas12b for human genome editing

Author

Listed:
  • Jonathan Strecker

    (Howard Hughes Medical Institute
    Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research, Department of Biological Engineering, Massachusetts Institute of Technology
    Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology)

  • Sara Jones

    (Howard Hughes Medical Institute
    Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research, Department of Biological Engineering, Massachusetts Institute of Technology
    Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology)

  • Balwina Koopal

    (Howard Hughes Medical Institute
    Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research, Department of Biological Engineering, Massachusetts Institute of Technology
    Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology)

  • Jonathan Schmid-Burgk

    (Howard Hughes Medical Institute
    Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research, Department of Biological Engineering, Massachusetts Institute of Technology
    Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology)

  • Bernd Zetsche

    (Howard Hughes Medical Institute
    Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research, Department of Biological Engineering, Massachusetts Institute of Technology
    Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology)

  • Linyi Gao

    (Howard Hughes Medical Institute
    Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research, Department of Biological Engineering, Massachusetts Institute of Technology
    Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology)

  • Kira S. Makarova

    (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health)

  • Eugene V. Koonin

    (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health)

  • Feng Zhang

    (Howard Hughes Medical Institute
    Broad Institute of MIT and Harvard
    McGovern Institute for Brain Research, Department of Biological Engineering, Massachusetts Institute of Technology
    Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology)

Abstract

The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. Here we explore the diversity of the Cas12b family and identify a promising candidate for human gene editing from Bacillus hisashii, BhCas12b. However, at 37 °C, wild-type BhCas12b preferentially nicks the non-target DNA strand instead of forming a double strand break, leading to lower editing efficiency. Using a combination of approaches, we identify gain-of-function mutations for BhCas12b that overcome this limitation. Mutant BhCas12b facilitates robust genome editing in human cell lines and ex vivo in primary human T cells, and exhibits greater specificity compared to S. pyogenes Cas9. This work establishes a third RNA-guided nuclease platform, in addition to Cas9 and Cpf1/Cas12a, for genome editing in human cells.

Suggested Citation

  • Jonathan Strecker & Sara Jones & Balwina Koopal & Jonathan Schmid-Burgk & Bernd Zetsche & Linyi Gao & Kira S. Makarova & Eugene V. Koonin & Feng Zhang, 2019. "Engineering of CRISPR-Cas12b for human genome editing," Nature Communications, Nature, vol. 10(1), pages 1-8, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-018-08224-4
    DOI: 10.1038/s41467-018-08224-4
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    Cited by:

    1. Xiangfeng Kong & Hainan Zhang & Guoling Li & Zikang Wang & Xuqiang Kong & Lecong Wang & Mingxing Xue & Weihong Zhang & Yao Wang & Jiajia Lin & Jingxing Zhou & Xiaowen Shen & Yinghui Wei & Na Zhong & W, 2023. "Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    2. Greta Bigelyte & Joshua K. Young & Tautvydas Karvelis & Karolina Budre & Rimante Zedaveinyte & Vesna Djukanovic & Elizabeth Ginkel & Sushmitha Paulraj & Stephen Gasior & Spencer Jones & Lanie Feigenbu, 2021. "Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells," Nature Communications, Nature, vol. 12(1), pages 1-8, December.
    3. Colin McGaw & Anthony J. Garrity & Gabrielle Z. Munoz & Jeffrey R. Haswell & Sejuti Sengupta & Elise Keston-Smith & Pratyusha Hunnewell & Alexa Ornstein & Mishti Bose & Quinton Wessells & Noah Jakimo , 2022. "Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    4. Marcus A. Toral & Carsten T. Charlesworth & Benjamin Ng & Teja Chemudupati & Shota Homma & Hiromitsu Nakauchi & Alexander G. Bassuk & Matthew H. Porteus & Vinit B. Mahajan, 2022. "Investigation of Cas9 antibodies in the human eye," Nature Communications, Nature, vol. 13(1), pages 1-9, December.

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