Multi-Pathway DNA Double-Strand Break Repair Reporters Reveal Extensive Cross-Talk Between End-Joining, Single Strand Annealing, and Homologous Recombination

Abstract

DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to extensive mutagenesis and genomic loss. Repair pathway cross-talk and compensation within the DSB-repair network is incompletely understood, despite its importance for genomic stability, oncogenesis, and the outcome of genome editing by CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple distinct pathways to repair of a DSB. These 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 inhibition of the essential c-NHEJ factor DNA-PKcs not only increases repair by HR, but also results in a substantial increase in mutagenic repair by SSA. We show that SSA-mediated repair of Cas9-generated DSBs can occur between Alu elements at endogenous genomic loci, and is enhanced by inhibition of DNA-PKcs. Finally, we demonstrate that the short-range end-resection factors CtIP and Mre11 promote both SSA and HR, whereas the long-range end-resection factors DNA2 and Exo1 promote SSA, but reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the rapid and comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.

Genomic Reporter Constructs to Monitor Pathway-Specific Repair of DNA Double-Strand Breaks

“…The BFP and GFP genes are completely homologous with exception of two amino acids that determine their fluorescent properties, and can therefore function as reciprocal repair templates without the need for additional homologous sequences. BFP to GFP conversion is also used as read-out for HR in the DSB-Spectrum reporters ( van de Kooij et al, 2021 ). Conveniently, no ectopic repair template is required in these multi-pathway reporter systems because the truncated GFP repair template is located on the same construct, downstream of the BFP gene that is targeted by Cas9.…”

“…The more recently developed DSB-Spectrum_V3 consists of an intact BFP gene that is targeted by Cas9, and can be disrupted by end-joining mediated mutagenesis. Loss of BFP expression is thus a measure for mutagenic end-joining in general, but primarily for mutagenic c-NHEJ, as indicated by sequence analysis ( van de Kooij et al, 2021 ). Furthermore, the reporter contains a truncated GFP gene that can be used to convert BFP to GFP as a measurement for HR ( van de Kooij et al, 2021 ).…”

“…Loss of BFP expression is thus a measure for mutagenic end-joining in general, but primarily for mutagenic c-NHEJ, as indicated by sequence analysis ( van de Kooij et al, 2021 ). Furthermore, the reporter contains a truncated GFP gene that can be used to convert BFP to GFP as a measurement for HR ( van de Kooij et al, 2021 ). These homologous elements can also anneal during DSB-repair by SSA, resulting in the removal of a functional mCherry gene that separates the two.…”