a, Schematic of two prominent DNA DSB repair pathways. A DSB can be repaired through various pathways that produce different DNA sequence end products. The NHEJ pathway is the dominant DSB repair pathway in most cells. The MMEJ pathway uses end-resection to discover small homologies on each side of the break that can be used to template the fusion of the broken ends. PARP-1 regulates DSB flux through the MMEJ pathway. Treatment of cells with rucaparib—an inhibitor of PARP-1—attenuates DSB flux down the MMEJ repair pathway. b, Percentage of microhomology-mediated deletions (green) and total indels (blue) resulting from SpCas9 treatment of cells in the presence of 0,10 and 20 μM rucaparib. Bars show mean and dots show individual data points from three biological replicates based on UMI-based Illumina deep sequencing. c, Percentage of 1-bp insertions (purple), microhomology mediated deletions (green) and other deletions (grey) produced by SpCas9 RNP with a sgRNA targeting the AAVS1 locus with the addition of increasing amounts of rucaparib. Mean ± s.e.m. from three biological replicates based on UMI-based Illumina deep sequencing. d, Percentage of microhomology-mediated deletions out of total indels in cells treated with SpCas9 in the presence of rucaparib. Mean ± s.e.m., dots represent individual data points from three biological replicates. P values determined using two-tailed unpaired t-test (Supplementary Table 9). ***P = 0.0004, ****P = 6.5 × 10−7. e, Left, alignment of allele sequences obtained from deep sequencing analysis from samples treated with SpCas9 RNP in the presence of different rucaparib concentrations. Microhomologies present at the AAVS1 locus are shown in by red, green and blue. Microhomology-mediated deletion is indicated by two-toned text. Magenta carets indicate site of DSB created by SpCas9. Inserted bases (ins) are shown in purple, deleted bases (del) are shown as black dashes. Right, heat map depicting the percentage of alleles generated after SpCas9 treatment of cells in the presence of different concentrations of rucaparib (0, 10 or 20 μM). The blue colour gradient scale indicates the percentage of occurrence of that sequence. Heat map represents mean values from a total of three independent biological replicates. Source data