A scalable CRISPR/Cas9-based fluorescent reporter assay to study DNA double-strand break repair choice. Color Assay Tracing Repair

DNA double-stranded breaks (DSBs) are the most toxic types of DNA lesions. Cells have evolved several strategies to repair such lesions that can be grouped into end-protection and end-resection coupled mechanisms. To study choices in DNA double-strand break (DSB) repair, we developed a color assay tracing repair (CAT-R) to simultaneously measure outcomes of DSB repair via end-protection and end-resection pathways. CAT-R relies on CRISPR/Cas9 to introduce DSBs in a tandem fluorescent reporter, which can distinguish small insertions/deletions from large deletions. We demonstrate CAT-R applications in chemical and genetic screens. First, we used CAT-R to evaluate 21 different compounds targeting factors in the DNA damage response that are currently in clinical trials. Second, we examined how 417 different factors involved in DNA damage repair influence the choice between end-protection and end-resection. Finally, we show that impairing nucleotide excision repair favored error-free repair, providing an alternative way of increasing the rate of CRISPR/Cas9-based knock-ins by homology-directed repair. In summary, CAT-R is a versatile assay to assess choices in DSB repair in high throughput.