Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks

Bert van de Kooij, Alex Kruswick, Haico van Attikum () and Michael B. Yaffe ()
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Bert van de Kooij: Leiden University Medical Center
Alex Kruswick: Massachusetts Institute of Technology
Haico van Attikum: Leiden University Medical Center
Michael B. Yaffe: Massachusetts Institute of Technology

Nature Communications, 2022, vol. 13, issue 1, 1-17

Abstract: Abstract DNA 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.

Date: 2022
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DOI: 10.1038/s41467-022-32743-w

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