Fig. 3

Optimisation of individual knockout. A All cell lines were nucleofected with a GFP plasmid using multiple pulse programs. The program that yielded the best transfection efficiency (number of GFP + cells divided with number of live cells in the control) and morphology was selected for each cell line (DN-100 for GU-pBT-7 and FT3465, CA-133 for GU-pBT-19, DS-134 for GU-pBT-28, CM-150 for GU-pBT-58, DS-150 for FT3477). B SOX2 was knocked out in GU-pBT-7 through nucleofection of RNP with different amounts of Cas9 (constant gRNA-to-Cas9 ratio) and the DNA cleavage decreased with decreasing amounts. C SOX2 knockout demonstrates loss of protein (green nuclei indicate SOX2 staining; DAPI in blue as counterstain for all nuclei) compared to D the control, and E more cells lose the SOX2 protein with increasing amounts. The control has significantly (p value < 0.05; Welch one-sided t test; indicated by *; n = 3 technical replicates for all conditions) higher proportion than all knockout condition. Knockout with 13 pmol Cas9 has significantly higher proportion of cells with retained SOX2 compared to 26 and 52 pmol. The error bars indicate standard error mean. F The SOX2 knockout cells and control cells were cultured for one month and counted at each split. The SOX2 knockout grew significantly slower than the control (p value < 0.05; Welch one-sided t test)