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CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome

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  • Ha Youn Shin

    (Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health
    Konkuk University)

  • Chaochen Wang

    (Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health)

  • Hye Kyung Lee

    (Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health
    Seoul National University)

  • Kyung Hyun Yoo

    (Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health
    Sookmyung Women’s University)

  • Xianke Zeng

    (Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health)

  • Tyler Kuhns

    (Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health)

  • Chul Min Yang

    (Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health)

  • Teresa Mohr

    (Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health)

  • Chengyu Liu

    (Transgenic Core, National Heart, Lung, and Blood Institute, US National Institutes of Health)

  • Lothar Hennighausen

    (Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health)

Abstract

Although CRISPR/Cas9 genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Here we interrogate the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome. We sequence targeted sites in 632 founder mice and analyse 54 established lines. While the median deletion size using single sgRNAs is 9 bp, we also obtain large deletions of up to 600 bp. Furthermore, we show unreported asymmetric deletions and large insertions of middle repetitive sequences. Simultaneous targeting of distant loci results in the removal of the intervening sequences. Reliable deletion of juxtaposed sites is only achieved through two-step targeting. Our findings also demonstrate that an extended analysis of F1 genotypes is required to obtain conclusive information on the exact molecular consequences of targeting events.

Suggested Citation

  • Ha Youn Shin & Chaochen Wang & Hye Kyung Lee & Kyung Hyun Yoo & Xianke Zeng & Tyler Kuhns & Chul Min Yang & Teresa Mohr & Chengyu Liu & Lothar Hennighausen, 2017. "CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome," Nature Communications, Nature, vol. 8(1), pages 1-10, August.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_ncomms15464
    DOI: 10.1038/ncomms15464
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    Cited by:

    1. Hye Kyung Lee & Michaela Willi & Chengyu Liu & Lothar Hennighausen, 2023. "Cell-specific and shared regulatory elements control a multigene locus active in mammary and salivary glands," Nature Communications, Nature, vol. 14(1), pages 1-17, December.
    2. Jianhang Yin & Kailun Fang & Yanxia Gao & Liqiong Ou & Shaopeng Yuan & Changchang Xin & Weiwei Wu & Wei-wei Wu & Jiaxu Hong & Hui Yang & Jiazhi Hu, 2022. "Safeguarding genome integrity during gene-editing therapy in a mouse model of age-related macular degeneration," Nature Communications, Nature, vol. 13(1), pages 1-8, December.
    3. Burcu Bestas & Sandra Wimberger & Dmitrii Degtev & Alexandra Madsen & Antje K. Rottner & Fredrik Karlsson & Sergey Naumenko & Megan Callahan & Julia Liz Touza & Margherita Francescatto & Carl Ivar Möl, 2023. "A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    4. Michael Kosicki & Felicity Allen & Frances Steward & Kärt Tomberg & Yangyang Pan & Allan Bradley, 2022. "Cas9-induced large deletions and small indels are controlled in a convergent fashion," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    5. G. Cullot & J. Boutin & S. Fayet & F. Prat & J. Rosier & D. Cappellen & I. Lamrissi & P. Pennamen & J. Bouron & S. Amintas & C. Thibault & I. Moranvillier & E. Laharanne & J. P. Merlio & V. Guyonnet-D, 2023. "Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9," Nature Communications, Nature, vol. 14(1), pages 1-16, December.

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