Fig. 6.

Targeting of endogenous loci in hESCs. a Gene targeting efficiency at the CALD1 (brown) and HIST1H2BK (yellow) loci in hESCs pES12 transfected with a CALD1 ssODN or a HIST1H2BK-mAG plasmid donor, respectively, along with vectors expressing Cas9 and sgRNA, and either a plasmid expressing e18 or an empty vector control. Gene targeting efficiency at the CALD1 and HIST1H2BK loci was determined by NGS and flow cytometry (mAG⁺ cells), respectively. Experiments were performed 5 days after transfection. HDR values are represented as in Fig. 5b (HIST1H2BK, n = 5; CALD1, n = 3). b Percentage of viable mAG⁺ hESC pES12 clones 14 days after transfection with Cas9/sgRNA-expressing vectors, mAG dsDNA donor targeting the HIST1H2BK locus, and either an empty or e18-expressing plasmid. The values of individual experiments are presented along with the mean ± s.e.m. (n = 3). Statistical significance was calculated using a paired t-test. c Immunostaining of pES12 cells edited at the HIST1H2BK locus using mAG dsDNA donors with or without e18 expression, as shown in a. Cell were stained using antibodies against the pluripotency markers NANOG (red) and SSEA-4 (cyan). mAG-tagged H2B is shown in green. Hoechst was used to detect DNA nuclei (blue). Merged images are also shown. Experiments were performed 5 days after transfection. Scale bar: 50 μm. Source data are available in the Source Data file