Fig. 3

High occurrence of backbone integration in all CRISPR–Cas9 mediated KI strategies. A Quantification of GFP expressing cells 2 days after transfection. GFP can only be expressed when the donor plasmid is integrated into the genome downstream of a transcribed RNA splice donor site. HEK293T cells were transfected with Cas9 and sgRNA plasmids along with the donor plasmid for either the unmatched ends, matched ends, long HA, or short HA strategy. Plasmid donor only transfections were performed as a control for random plasmid integration into the genome. Error bars indicate standard error from 3 biological replicates. B Fluorescence profile of puromycin-selected cells. Transfected cells were selected with puromycin for 3 weeks prior to Flow cytometry analysis. Any BFP expression arising from the backbone of the transiently transfected plasmid is lost by this time, so the relative proportions of cells expressing GFP and/or BFP can be calculated. The percentages of green, blue, and double fluorescent cells are plotted as stacked graphs. Error bars indicates standard error for GFP+ cells from three biological replicates. *p < 0.05 in student T-test