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Massively targeted evaluation of therapeutic CRISPR off-targets in cells

Author

Listed:
  • Xiaoguang Pan

    (Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
    Copenhagen University)

  • Kunli Qu

    (Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
    Copenhagen University
    Aarhus University)

  • Hao Yuan

    (Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
    University of Chinese Academy of Sciences)

  • Xi Xiang

    (Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
    Aarhus University)

  • Christian Anthon

    (University of Copenhagen)

  • Liubov Pashkova

    (University of Copenhagen)

  • Xue Liang

    (Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
    Copenhagen University)

  • Peng Han

    (Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
    Copenhagen University)

  • Giulia I. Corsi

    (University of Copenhagen)

  • Fengping Xu

    (Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
    University of Chinese Academy of Sciences
    BGI-Research, BGI-Shenzhen)

  • Ping Liu

    (BGI-Research, BGI-Shenzhen
    MGI, BGI-Shenzhen)

  • Jiayan Zhong

    (BGI-Research, BGI-Shenzhen
    MGI, BGI-Shenzhen)

  • Yan Zhou

    (Aarhus University)

  • Tao Ma

    (BGI-Research, BGI-Shenzhen
    MGI, BGI-Shenzhen)

  • Hui Jiang

    (BGI-Research, BGI-Shenzhen
    MGI, BGI-Shenzhen)

  • Junnian Liu

    (Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen)

  • Jian Wang

    (BGI-Research, BGI-Shenzhen)

  • Niels Jessen

    (Aarhus University
    Aarhus University Hospital)

  • Lars Bolund

    (Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
    Aarhus University)

  • Huanming Yang

    (BGI-Research, BGI-Shenzhen
    Chinese Academy of Sciences)

  • Xun Xu

    (BGI-Research, BGI-Shenzhen
    Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen)

  • George M. Church

    (Harvard Medical School)

  • Jan Gorodkin

    (University of Copenhagen)

  • Lin Lin

    (Aarhus University
    Aarhus University Hospital)

  • Yonglun Luo

    (Lars Bolund Institute of Regenerative Medicine, Qingdao-Europe Advanced Institute for Life Sciences, BGI-Qingdao, BGI-Shenzhen
    Aarhus University
    University of Chinese Academy of Sciences
    BGI-Research, BGI-Shenzhen)

Abstract

Methods for sensitive and high-throughput evaluation of CRISPR RNA-guided nucleases (RGNs) off-targets (OTs) are essential for advancing RGN-based gene therapies. Here we report SURRO-seq for simultaneously evaluating thousands of therapeutic RGN OTs in cells. SURRO-seq captures RGN-induced indels in cells by pooled lentiviral OTs libraries and deep sequencing, an approach comparable and complementary to OTs detection by T7 endonuclease 1, GUIDE-seq, and CIRCLE-seq. Application of SURRO-seq to 8150 OTs from 110 therapeutic RGNs identifies significantly detectable indels in 783 OTs, of which 37 OTs are found in cancer genes and 23 OTs are further validated in five human cell lines by targeted amplicon sequencing. Finally, SURRO-seq reveals that thermodynamically stable wobble base pair (rG•dT) and free binding energy strongly affect RGN specificity. Our study emphasizes the necessity of thoroughly evaluating therapeutic RGN OTs to minimize inevitable off-target effects.

Suggested Citation

  • Xiaoguang Pan & Kunli Qu & Hao Yuan & Xi Xiang & Christian Anthon & Liubov Pashkova & Xue Liang & Peng Han & Giulia I. Corsi & Fengping Xu & Ping Liu & Jiayan Zhong & Yan Zhou & Tao Ma & Hui Jiang & J, 2022. "Massively targeted evaluation of therapeutic CRISPR off-targets in cells," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-31543-6
    DOI: 10.1038/s41467-022-31543-6
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    Cited by:

    1. Jianli Tao & Daniel E. Bauer & Roberto Chiarle, 2023. "Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    2. Qinchang Chen & Guohui Chuai & Haihang Zhang & Jin Tang & Liwen Duan & Huan Guan & Wenhui Li & Wannian Li & Jiaying Wen & Erwei Zuo & Qing Zhang & Qi Liu, 2023. "Genome-wide CRISPR off-target prediction and optimization using RNA-DNA interaction fingerprints," Nature Communications, Nature, vol. 14(1), pages 1-17, December.

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