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

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

  • Nat Commun. 2022 Sep 8;13(1):5295. doi: 10.1038/s41467-022-32743-w.
Bert van de Kooij 1 2,  Alex Kruswick 2,  Haico van Attikum 3,  Michael B Yaffe 4 5 6
Affiliations

Affiliations

  • 1 Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, the Netherlands.
  • 2 Koch Institute for Integrative Cancer Research, MIT Center for Precision Cancer Medicine, Departments of Biology and Bioengineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
  • 3 Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, the Netherlands. H.van.Attikum@lumc.nl.
  • 4 Koch Institute for Integrative Cancer Research, MIT Center for Precision Cancer Medicine, Departments of Biology and Bioengineering, Massachusetts Institute of Technology, Cambridge, MA, USA. myaffe@mit.edu.
  • 5 Department of Surgery, Beth Israel Deaconess Medical Center, Divisions of Acute Care Surgery, Trauma, and Critical Care and Surgical Oncology, Harvard Medical School, Boston, MA, USA. myaffe@mit.edu.
  • 6 Surgical Oncology Program, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. myaffe@mit.edu.
PMID: 36075911 DOI: 10.1038/s41467-022-32743-w
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.

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