Fig. 3

Selected Cas9-expressing single cell clones show stronger editing efficiency compared to a Cas9 bulk population. a Workflow for the selection of Cas9 single-cell clones (SCCs). SCCs were sorted from the HAP1 Cas9 bulk population and further characterized. Cas9 editing was assessed by cell surface marker knockout followed by FACS staining and cell viability upon knockout of a core essential gene. Two highly editing single-cell clones (SCC11 and SCC12) were selected for further experiments. b HAP1 Cas9 bulk, Cas9 SCC11, and Cas9 SCC12 cells were transfected with the HDCRISPRv1 vector encoding an sgRNA targeting either the safe harbor locus AAVS1 as a control or the core essential gene RNA Polymerase 2 subunit E (POLR2E). Editing efficiency based on cell viability of sgPOLR2E-transfected cells in comparison to sgAAVS1 control cells was addressed by crystal violet staining. The number of surviving cells was strongly reduced in cells transfected with an sgRNA directed against POLR2E (n = 3 for each cell line and sgRNA). c Editing efficiency was furthermore assessed upon transduction of HAP1 Cas9 bulk, Cas9 SCC11, and Cas9 SCC12 cells with the HDCRISPRv1 vector expressing sgRNAs targeting the surface marker CD46, followed by FACS staining of residual CD46 protein to address knockout efficiency. Antibody staining of the non-edited cell lines was used as a control. Lines represent the mean of independent measurements (n = 3 for each cell line and condition)