Fig. 3
Validation of a reporter-less NHEJ assay in cells deficient for XRCC4. a Deletion of the intervening sequence between two target sites by paired Cas9-gRNA. Cells were transfected with expression plasmids for single or paired sgRNAs and Cas9 and genomic DNA was purified 72 h post-transfection and amplified by primers flanking the cutting sites. The PCR amplicons were subjected to agarose gel electrophoresis. The distance between two cleavage sites was 57 bp at the LDHA site and 33 bp at the ROSA26 site as indicated and the deletion of the intervening sequence indicates simultaneous Cas9 cleavage. b–d Analysis of NHEJ induced by paired Cas9-gRNA at the LDHA and ROSA26 sites in isogenic XRCC4+/+ and XRCC4−/− mouse ES cells. The normalized editing efficiency (b), the frequency of each group in edited events (c), and the frequency of each category in group I events (d) were calculated. The normalized editing efficiency represents the efficiency of overall NHEJ, including accurate and mutagenic NHEJ. Bars represent the mean ± standard deviation (SD) of three independent experiments. For normalized editing efficiency (b), Student’s paired t-test between XRCC4+/+ and XRCC4−/− P = 0.0028 at the LDHA site. e Deletion length distributions of Group I ‘Del’ events in isogenic XRCC4+/+ and XRCC4−/− mouse ES cells. The reads were combined by three independent experiments. At the LDHA site and the ROSA26 site, each dot represents 100 reads and 20 reads, respectively. The median deletion length is indicated, and deletion distributions demonstrate a shift towards longer deletions in cells lacking XRCC4 (Mann–Whitney test, ****P < 0.0001). f Frequency of accurate NHEJ among group I (left) and frequency of deletions with different deletion length in “Del” events of group I NHEJ (right) in either isogenic XRCC4+/+ or XRCC4−/− mouse ES cells. Del NHEJ events were grouped into 58–63 bp and > 63 bp at the LDHA site and 34–39 bp and > 39 bp at the ROSA26 site. The respective reads and frequencies were summarized in the inset and compared by a χ2 test with P values indicated. g Frequency of microhomology usage at different lengths in deletion-only group I events of either isogenic XRCC4+/+ or XRCC4−/− mouse ES cells