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Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair

Author

Listed:
  • Beeke Wienert

    (University of California
    University of California
    Gladstone Institutes)

  • David N. Nguyen

    (University of California
    University of California
    University of California)

  • Alexis Guenther

    (University of California)

  • Sharon J. Feng

    (University of California
    University of California)

  • Melissa N. Locke

    (University of California)

  • Stacia K. Wyman

    (University of California)

  • Jiyung Shin

    (ETH Zürich)

  • Katelynn R. Kazane

    (University of California
    University of California)

  • Georgia L. Gregory

    (Gladstone Institutes)

  • Matthew A. M. Carter

    (Gladstone Institutes)

  • Francis Wright

    (University of California)

  • Bruce R. Conklin

    (Gladstone Institutes
    University of California)

  • Alex Marson

    (University of California
    University of California
    University of California
    University of California)

  • Chris D. Richardson

    (University of California
    University of California
    University of California)

  • Jacob E. Corn

    (University of California
    University of California
    ETH Zürich)

Abstract

Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification.

Suggested Citation

  • Beeke Wienert & David N. Nguyen & Alexis Guenther & Sharon J. Feng & Melissa N. Locke & Stacia K. Wyman & Jiyung Shin & Katelynn R. Kazane & Georgia L. Gregory & Matthew A. M. Carter & Francis Wright , 2020. "Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair," Nature Communications, Nature, vol. 11(1), pages 1-15, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-15845-1
    DOI: 10.1038/s41467-020-15845-1
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    Cited by:

    1. Lorenzo Galanti & Martina Peritore & Robert Gnügge & Elda Cannavo & Johannes Heipke & Maria Dilia Palumbieri & Barbara Steigenberger & Lorraine S. Symington & Petr Cejka & Boris Pfander, 2024. "Dbf4-dependent kinase promotes cell cycle controlled resection of DNA double-strand breaks and repair by homologous recombination," Nature Communications, Nature, vol. 15(1), pages 1-19, December.
    2. Lukas Möller & Eric J. Aird & Markus S. Schröder & Lena Kobel & Lucas Kissling & Lilly van de Venn & Jacob E. Corn, 2022. "Recursive Editing improves homology-directed repair through retargeting of undesired outcomes," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    3. Zsolt Bodai & Alena L. Bishop & Valentino M. Gantz & Alexis C. Komor, 2022. "Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies," Nature Communications, Nature, vol. 13(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.

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