Supplementary Figure 7: Improvement of first-round editing by shRNA-based removal of random integrant and PCR-based copy-number analysis using modified UBC promoter in B2M editing.

(a, b) Schematic representation of donor DNA design and genotyping by PCR in B2M editing of the H9 hESC line. (a) First-round editing without shGFP expression cassette. (b) First-round editing with shGFP expression cassette. All clones shown in a and b expressed bright GFP fluorescence. (c) GG>AA mutation at 314th and 315th upstream from ATG start codon. In B2M editing, modified UBC promoter (GG>AA) was used for selection marker expression. (d) Flow cytometry analysis showing WT and GG>AA mutant of UBC promoter expressed at almost the same intensity of GFP in K562 cells. (e) Schematic representation of primer design and size of PCR amplicon after EcoRI digestion. (f) Agarose gel electrophoresis of PCR products digested with EcoRI-HF enzyme. DNA was stained with MidoriGreen dye. (g) Representative image from image analysis by ImageJ software. (h) Quantification of fluorescence intensity (FI) in samples from regular PCR combined with EcoRI digestion. All samples from bright clones show the same intensity between WT and GG>AA, meaning two copies of exogenous UBC promoter were integrated, and FIs of GG>AA were half of those of the WT in samples from dim clones, meaning one copy of exogenous UBC promoter was integrated. (i) Copy-number analysis of UBC promoter via a ddPCR-based method. The result from ddPCR was consistent with that of the regular PCR-EcoRI digestion–based experiment. In panels h and i, individual blue points represent technical replicates (n = 3) and orange bars represent the mean value. FCM and PCR shown here were repeated twice, with similar results each time.