Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks
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s41467-022-32743-w.pdf
Description
Published version
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2.19 MB
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Author(s)
van de Kooij, Bert
Kruswick, Alex
van Attikum, Haico
Yaffe, Michael B
Date Issued
September 8, 2022
Journal
Nature Communications
Publisher
Springer Science and Business Media LLC
Citation
van de Kooij, Bert, Kruswick, Alex, van Attikum, Haico and Yaffe, Michael B. 2022. "Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks." Nature Communications, 13 (1).
Version
Final published version
Abstract
AbstractDNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to mutagenesis and genomic loss. DSB-repair pathway cross-talk and compensation is incompletely understood, despite its importance for genomic stability, oncogenesis, and genome editing using CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple DNA repair pathways at a DSB. DSB-Spectrum reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibiting the c-NHEJ factor DNA-PKcs increases repair by HR, but also substantially increases mutagenic SSA. Our data indicate that SSA-mediated DSB-repair also occurs at endogenous genomic loci, driven by Alu elements or homologous gene regions. Finally, we demonstrate that long-range end-resection factors DNA2 and Exo1 promote SSA and reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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Creative Commons Attribution 4.0 International license
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DOI of Published Version
https://doi.org/10.1038/s41467-022-32743-w
