Fig. 4: SSA suppression reduces asymmetric HDR repair in the HDR reporter assay.

a Overview of the HDR reporter system. The HDR reporter cassette is monoallelically inserted at the immediate upstream of the stop codon of HNRNPA1 in RPE1 cells. The seamless, perfect HDR repair of the Cas9-induced DSBs in the reporter cassette completes the two split fluorescent protein sequences, resulting in their simultaneous fluorescence emission (Double positive). The presence of either mNG or mScarlet fluorescence alone (Single positive) shows asymmetric HDR repair with precise editing only at the 3’ or 5’ end, respectively. The sequence of the donor DNA is shown in Supplementary Table 3. b Representative images of the HDR reporter cell line exhibiting both SNAP-tag and HaloTag ligands signals in the localization corresponding to the nuclear protein HNRNPA1. The parental wild type (WT) cells were analyzed as a control. Scale bar: 10 µm. c Time course of the HDR reporter assay and a representative plot obtained through flow cytometric analysis. After electroporation, the cells were treated with DMSO or NHEJi for 24 h. The plot displays the percentages of cells exhibiting only mNG signal (green), only mScarlet signal (magenta), or both signals (orange). d The ratio of the single-positive cells to double-positive cells in the HDR reporter assay. Cells were treated with the indicated inhibitors for 24 h after electroporation. Data from three biological replicates are presented as mean ± S.D. and a two-tailed, unpaired Student’s t test was used to obtain the P-value. *P < 0.05.