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CRISPR-Cas3 induces broad and unidirectional genome editing in human cells

Author

Listed:
  • Hiroyuki Morisaka

    (Osaka University
    Kochi University)

  • Kazuto Yoshimi

    (Osaka University
    Graduate School of Medicine, Osaka University
    The University of Tokyo)

  • Yuya Okuzaki

    (Kyoto University)

  • Peter Gee

    (Kyoto University)

  • Yayoi Kunihiro

    (Osaka University)

  • Ekasit Sonpho

    (Graduate School of Medicine, Osaka University
    Faculty of Science, Mahidol University)

  • Huaigeng Xu

    (Kyoto University)

  • Noriko Sasakawa

    (Kyoto University)

  • Yuki Naito

    (Database Center for Life Science
    National Institute of Genetics)

  • Shinichiro Nakada

    (Osaka University)

  • Takashi Yamamoto

    (Hiroshima University)

  • Shigetoshi Sano

    (Kochi University)

  • Akitsu Hotta

    (Kyoto University)

  • Junji Takeda

    (Osaka University
    Osaka University)

  • Tomoji Mashimo

    (Osaka University
    Graduate School of Medicine, Osaka University
    The University of Tokyo)

Abstract

Although single-component Class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5′-ARG protospacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system.

Suggested Citation

  • Hiroyuki Morisaka & Kazuto Yoshimi & Yuya Okuzaki & Peter Gee & Yayoi Kunihiro & Ekasit Sonpho & Huaigeng Xu & Noriko Sasakawa & Yuki Naito & Shinichiro Nakada & Takashi Yamamoto & Shigetoshi Sano & A, 2019. "CRISPR-Cas3 induces broad and unidirectional genome editing in human cells," Nature Communications, Nature, vol. 10(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-13226-x
    DOI: 10.1038/s41467-019-13226-x
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    Cited by:

    1. Anna Zimmermann & Julian E. Prieto-Vivas & Charlotte Cautereels & Anton Gorkovskiy & Jan Steensels & Yves Peer & Kevin J. Verstrepen, 2023. "A Cas3-base editing tool for targetable in vivo mutagenesis," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    2. Meiling Lu & Chenlin Yu & Yuwen Zhang & Wenjun Ju & Zhi Ye & Chenyang Hua & Jinze Mao & Chunyi Hu & Zhenhuang Yang & Yibei Xiao, 2024. "Structure and genome editing of type I-B CRISPR-Cas," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    3. Jing Guo & Luyao Gong & Haiying Yu & Ming Li & Qiaohui An & Zhenquan Liu & Shuru Fan & Changjialian Yang & Dahe Zhao & Jing Han & Hua Xiang, 2024. "Engineered minimal type I CRISPR-Cas system for transcriptional activation and base editing in human cells," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    4. Duško Lainšček & Vida Forstnerič & Veronika Mikolič & Špela Malenšek & Peter Pečan & Mojca Benčina & Matjaž Sever & Helena Podgornik & Roman Jerala, 2022. "Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing," Nature Communications, Nature, vol. 13(1), pages 1-12, December.

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