Fig. 6
From: Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones
CRISPR/Cas9 base edition and single cell-derived clone characterization. A Schematic representation of the region (exon 16 in ALMS1 gene) targeted by CRISPR/Cas9 with the selected gRNA. B SANGER sequences of the site of edition before CRISPR/Cas9 base edition (WT before selection; upper panel), following edition in a representative edited cell population (pooled cells; middle panel), and in a representative single cell-derived clone using the semi-automated method (lower panel). The gRNA is underlined in black and the PAM motif in red. The dotted rectangle corresponds to the position targeted for base editing. C Immunofluorescence assay using pluripotency markers (OCT3/4 and LIN28A) on WT before selection and p.Gln3613Ter purified hPSC clone (lower panel). Scale bar = 50 µm. D, E Evaluation of pluripotency of p.Gln3613Ter and p.Glu192fs hPSCs by flow cytometry (pluripotency markers OCT3/4 and LIN28A (D) and TRA1-81 and SSEA-4 (E)). F Immunofluorescence assay showing the expression of Pericentrin (red) and ALMS1 (green) on WT (upper panel) and p.Gln3613Ter purified hPSC clone (lower). Scale bar = 5 µm. G Quantification of ALMS1 area (µm2) co-localized with Pericentrin according to the genotype (wild type, p.Glu192fs, p.Gln3613Ter). H Karyotype of the p.Gln3613Ter purified hPSC clone