Fig. 6

Targeted in-frame deletions mediated by accurate NHEJ assists functional domain analysis in situ. a Correlation between accurate NHEJ and in-frame deletions generated by the Common (black triangles), Ideal (blue squares), and Paired approaches (red circles). b Domain structure of mouse 53BP1 protein. Oligomerization domain (OD), Tudor domain, nuclear localization sequence (NLS), and tandem BRCT domain are shown. Two sets of paired gRNAs respectively targeting the Tudor domain (exon 21) and OD domain (exon 18) are also indicated. Precise in-frame deletions were confirmed by Sanger sequencing of the PCR product for repair junction. c Detection of 53BP1 wild-type and in-frame mutants by western blotting using β-actin as loading control. WT wild type. d IR-induced foci formation of γH2AX (red) and 53BP1 (green) in 53BP1 WT clone and in-frame mutant clones. e–g Analysis of NHEJ induced by paired Cas9-gRNAs at the LDHA site in 53BP1 WT clone and in-frame mutant clones. The normalized editing efficiency (e), the frequency of each group in edited events (f), and the frequency of each category in group I events (g) were calculated. Bars represent the mean ± standard deviation (SD) of three independent experiments. h Deletion length distributions of group I “Del” events at the LDHA site in 53BP1 WT mouse ES clone and in-frame mutant clones. The reads were combined from three independent experiments. Each dot represents 20 reads. The median deletion length is indicated, and deletion length distributions demonstrate a shift towards longer deletions in 53BP1 in-frame mutant clones (Mann–Whitney test between WT and ΔTudor or ΔOD ****P < 0.0001). i 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 53BP1 WT mouse ES clone and in-frame mutant clones. Del NHEJ events were grouped into 58–63 bp and > 63 bp. The respective reads and frequencies are summarized in the inset and compared by a χ² test with P values as indicated