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CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations

Author

Listed:
  • Ida Höijer

    (Uppsala University)

  • Anastasia Emmanouilidou

    (Uppsala University
    Uppsala University)

  • Rebecka Östlund

    (Uppsala University)

  • Robin Schendel

    (Leiden University Medical Center)

  • Selma Bozorgpana

    (Uppsala University)

  • Marcel Tijsterman

    (Leiden University Medical Center)

  • Lars Feuk

    (Uppsala University)

  • Ulf Gyllensten

    (Uppsala University)

  • Marcel Hoed

    (Uppsala University
    Uppsala University)

  • Adam Ameur

    (Uppsala University)

Abstract

CRISPR-Cas9 genome editing has potential to cure diseases without current treatments, but therapies must be safe. Here we show that CRISPR-Cas9 editing can introduce unintended mutations in vivo, which are passed on to the next generation. By editing fertilized zebrafish eggs using four guide RNAs selected for off-target activity in vitro, followed by long-read sequencing of DNA from >1100 larvae, juvenile and adult fish across two generations, we find that structural variants (SVs), i.e., insertions and deletions ≥50 bp, represent 6% of editing outcomes in founder larvae. These SVs occur both at on-target and off-target sites. Our results also illustrate that adult founder zebrafish are mosaic in their germ cells, and that 26% of their offspring carries an off-target mutation and 9% an SV. Hence, pre-testing for off-target activity and SVs using patient material is advisable in clinical applications, to reduce the risk of unanticipated effects with potentially large implications.

Suggested Citation

  • Ida Höijer & Anastasia Emmanouilidou & Rebecka Östlund & Robin Schendel & Selma Bozorgpana & Marcel Tijsterman & Lars Feuk & Ulf Gyllensten & Marcel Hoed & Adam Ameur, 2022. "CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-28244-5
    DOI: 10.1038/s41467-022-28244-5
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    References listed on IDEAS

    as
    1. Xin Luo & Yaoxi He & Chao Zhang & Xiechao He & Lanzhen Yan & Min Li & Ting Hu & Yan Hu & Jin Jiang & Xiaoyu Meng & Weizhi Ji & Xudong Zhao & Ping Zheng & Shuhua Xu & Bing Su, 2019. "Trio deep-sequencing does not reveal unexpected off-target and on-target mutations in Cas9-edited rhesus monkeys," Nature Communications, Nature, vol. 10(1), pages 1-7, December.
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    Cited by:

    1. Hsiu-Hui Tsai & Hsiao-Jung Kao & Ming-Wei Kuo & Chin-Hsien Lin & Chun-Min Chang & Yi-Yin Chen & Hsiao-Huei Chen & Pui-Yan Kwok & Alice L. Yu & John Yu, 2023. "Whole genomic analysis reveals atypical non-homologous off-target large structural variants induced by CRISPR-Cas9-mediated genome editing," Nature Communications, Nature, vol. 14(1), pages 1-9, December.

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