Fig. 5: Enrichment of genome editing events in human iPSCs.
From: Universal toxin-based selection for precise genome engineering in human cells
a Schema of the experiment timeline. b, c Co-selection of CBE (b) and ABE (c) editing events in the genome of hiPSCs cultured with or without DT. Analysis of NGS reads showing containing b C–T conversions or c A–G conversions as a percentage of full-length reads from indicated loci using depicted sgRNAs. d Enrichment of knock-in events at HBEGF locus with Xential. The left panel shows the mCherry signal in the flow cytometry scatter plots for non-enriched and DT-enriched samples, and the right panel shows the quantitative frequencies of mCherry-positive cells. e PCR analysis of genomic DNA from hiPSCs after Xential. Genotyping at HBEGF intron 3 (see Fig. 3d). Representative results were shown from three independent biological replicates. f–g The HSV-TK gene inserted in the HBEGF locus using Xential renders hiPSCs sensitive to gancivlovir. Crystal violet staining assay (f) and CellTiter-Glo assay (g) showing viability of cells containing either the mCherry or the HSV-TK gene inserted in the HBEGF locus. Cells were treated with depicted concentration of ganciclovir or DMSO (control) for 3 days followed by a 3-day recovery without the drug. Values and error bars reflect mean ± s.d. of n = 3 independent biological replicates. Relative fold changes are indicated in the graphs. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s paired t test (two-tailed). P values are calculated as below: in b, CTLA4 (C4 = 0.0179 and C5 = 0.0007) and EMX1 (C5 = 0.0010 and C6 = 0.0006); in c, CTLA4 (0.0154) and EMX1 (0.1146). Source data of Fig. 5b–e, g are provided as a Source data file.
