Abstract
DNA double-strand breaks (DSBs) are highly cytotoxic lesions that lead to genomic instability, chromosome rearrangements, oncogenic transformation, and sensitivity to radio- and chemotherapies. The accurate and efficient repair of DSBs is critical for maintaining genomic integrity, and cells employ distinctive pathways to resolve these breaks. Elucidating the mechanisms underlying DSB repair not only increases our understanding of cancer etiology, but also helps identify novel molecular targets for therapeutic intervention.
We have developed a new reporter system, designated CDDR (CRISPR-Cas9-based dual-fluorescent DSB repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells. CDDR is based on the efficient introduction of one or two synchronous DSB(s) within an intrachromosomal fluorescent reporter via the co-expression of Cas9 and sgRNAs targeting the reporter. Quantitative flow cytometry combined with high-throughput sequencing of repair junctions reports on the repair of DSBs by non-homologous end-joining (NHEJ) and by homology-directed repair (HDR) with high efficiency and remarkable reproducibility. CDDR can discriminate between high-fidelity (HF) and mutagenic NHEJ, as well as between proximal (at a single DSB site) and distal (between two DSBs) NHEJ repair. Furthermore, CDDR can detect HDR at frequencies significantly higher than those detected by other HDR reporters.
Using the CDDR reporter system, we sought to investigate the role of a subset of repair proteins in the resolution of DSBs. Towards this goal, we generated isogenic cell lines deficient of these proteins in U2OS cells containing the CDDR reporter. Our results reveal novel functions for these repair factors and their contribution to the fidelity of DSB repair by NHEJ, the utilization of distal vs proximal ends in repairing DSBs, the use of microhomology in NHEJ-mediated deletions, as well as to the repair of DSBs via HDR. Using isogenic U2OS cells containing the CDDR reporter and with deletions of key repair proteins, we found proximal and distal HF-NHEJ to be dependent upon the classical NHEJ factors XLF, XRCC4 and DNA Ligase IV. The loss of these genes stimulated deletion mutagenesis and promoted distal end-utilization in the repair of DSBs. Furthermore, the deletion of these genes stimulated microhomology-mediated end-joining and HDR. Deletion of DNA-PKcs and 53BP1, which function upstream of XRCC4, XLF and LIG4 in the cNHEJ pathway, did not diminish HF-NHEJ to the same extent as other NHEJ factors, nor did it impact distal end-utilization. However, we did observe a significant increase in HDR in these cells, similar to that observed in cells deleted of XRCC4, XLF and LIG4. By contrast, we found that the loss of ATM or NBS1 stimulated HF-NHEJ and suppressed microhomology use and HDR.
These findings demonstrate the efficacy of the CDDR reporter system in the detection and quantification of DSB repair outcomes in mammalian cells with high sensitivity and reproducibility, and without the need for normalization steps. We also uncover distinct new features for a subset of repair proteins that provide insight into the balance between competing DSB repair pathways and extend our understanding of the contribution of these proteins to the resolution of DSBs.
