Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks

Eva K. Brinkman, Tao Chen, Marcel de Haas, Linda Holland, Waseem Akhtar, Bas van Steensel

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to ∼10 hr. Furthermore, repair of the DSBs tends to be error prone. Both classical and microhomology-mediated end joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci and indicates that Cas9-induced DSBs are repaired in an unusual manner. Brinkman et al. report a strategy to determine the rate and fidelity of double-strand break repair at single loci cut by Cas9. They also infer the contributions from different repair pathways. Cas9-induced breaks are repaired at variable but often slow rates, and the repair tends to be error prone.

Original languageEnglish
Pages (from-to)801-813.e1-e6
JournalMolecular Cell
Volume70
Issue number5
DOIs
Publication statusPublished - 2018
Externally publishedYes

Funding

We thank B. Evers for sharing plasmids prior to publication, Jeroen van den Berg for help with experiments, H. te Riele and members of our laboratory for critical reading of the manuscript, and the NKI Genomics and flow cytometry core facilities for technical assistance. This work was supported by a ZonMW-TOP grant ( 91211061 ) and ERC Advanced Grants 293662 and 694466 (to B.v.S.). W.A. was supported by an NWO-ALW grant awarded to M. van Lohuizen. Oncode Institute is supported by the Dutch Cancer Society (KWF).

FundersFunder number
Dutch Cancer Society
NWO-ALW
Horizon 2020 Framework Programme694466, 293662
European Research Council
KWF Kankerbestrijding

    Keywords

    • CRISPR-Cas
    • DNA double-strand break
    • genome editing
    • microhomology-mediated end-joining
    • non-homologous end-joining
    • repair kinetics

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