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Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus

Author

Listed:
  • Anne Bothmer

    (Editas Medicine)

  • Tanushree Phadke

    (Editas Medicine)

  • Luis A. Barrera

    (Editas Medicine)

  • Carrie M Margulies

    (Editas Medicine)

  • Christina S. Lee

    (Editas Medicine)

  • Frank Buquicchio

    (Editas Medicine)

  • Sean Moss

    (Editas Medicine)

  • Hayat S. Abdulkerim

    (Editas Medicine)

  • William Selleck

    (Editas Medicine)

  • Hariharan Jayaram

    (Editas Medicine)

  • Vic E. Myer

    (Editas Medicine)

  • Cecilia Cotta-Ramusino

    (Editas Medicine)

Abstract

The CRISPR–Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR–Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies.

Suggested Citation

  • Anne Bothmer & Tanushree Phadke & Luis A. Barrera & Carrie M Margulies & Christina S. Lee & Frank Buquicchio & Sean Moss & Hayat S. Abdulkerim & William Selleck & Hariharan Jayaram & Vic E. Myer & Cec, 2017. "Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus," Nature Communications, Nature, vol. 8(1), pages 1-12, April.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_ncomms13905
    DOI: 10.1038/ncomms13905
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    Cited by:

    1. Jun Huang & David Rowe & Pratima Subedi & Wei Zhang & Tyler Suelter & Barbara Valent & David E. Cook, 2022. "CRISPR-Cas12a induced DNA double-strand breaks are repaired by multiple pathways with different mutation profiles in Magnaporthe oryzae," Nature Communications, Nature, vol. 13(1), pages 1-18, December.
    2. Sébastien Levesque & Diana Mayorga & Jean-Philippe Fiset & Claudia Goupil & Alexis Duringer & Andréanne Loiselle & Eva Bouchard & Daniel Agudelo & Yannick Doyon, 2022. "Marker-free co-selection for successive rounds of prime editing in human cells," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    3. Aron Ferenczi & Yen Peng Chew & Erika Kroll & Charlotte Koppenfels & Andrew Hudson & Attila Molnar, 2021. "Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
    4. J. Ferreira da Silva & G. P. Oliveira & E. A. Arasa-Verge & C. Kagiou & A. Moretton & G. Timelthaler & J. Jiricny & J. I. Loizou, 2022. "Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair," Nature Communications, Nature, vol. 13(1), pages 1-11, December.

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