Extended Data Fig. 3: Pluripotency, trilineage differentiation and single cell cloning analysis of CCR5 gene targeted PSCs.
a. Percentage of cells positive for various markers of pluripotency (SSEA4, OCT3/4, SOX2 and NANOG) in the CCR5 gene edited PSCs (n = 3). SSEA4 expression was measured by flow cytometry analysis. OCT3/4, SOX2 and NANOG expression was assessed by quantification of immunofluorescence staining images for corresponding markers in fixed PSCs and normalized to the total cell count measured through DAPI staining (n = 3). All data are shown as mean ± SEM. b. CCR5 gene edited PSCs were differentiated into the three germ layers. Mean frequency of differentiated cells as assessed by flow cytometry for the expression of corresponding markers for ectoderm (PAX6 and NES), mesoderm (CD56 and T) and endoderm (CXCR4 and SOX17) (n = 2). c. PSCs were gene targeted at the CCR5 locus with and without different concentrations of AZD7648 (0.5, 0.25 and 0.1 µM) for single cell cloning analysis. Allelic gene targeting efficiency was measured using ddPCR analysis (n = 1). d. Gene targeted PSCs (c) were subjected to single cell cloning and the frequency of clones with mono-, bi-allelic and no gene targeting was measured using ddPCR and PCR analysis. For each condition, 9-10 clones were picked and analyzed (n = 1). e. Mean allelic gene targeting efficiency in PSC at the CCR5 locus for the knock-in of UBC-GFP-bGHpA sequence with pre-treatment only (preAZD-UNT), post-treatment (AZD) only and pre+post treatment (pre+post AZD) with AZD7648 (0.5 µM) (n = 2). For pre-treatment, cells were treated with AZD7648 for 24 hours before gene targeting and for post-treatment, cells were treated with AZD7648 for 24 hours post gene editing.
