Fig. 4
From: In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting
Competition for gene targeting between donor DNA resistant and sensitive to RGN-induced nicking. a Schematics of the experimental design. HeLa cells were co-transfected with the indicated donor templates together with plasmids encoding nicking Cas9D10A:gRNAS1. b Quantification of stably transfected cell populations. The frequencies of genetically modified cells were determined at 27 days post-transfection by EGFP-directed and mTurquoise2-directed flow cytometry. The ratios between the frequencies of the various gene-modified subpopulations are presented. c Flow cytometry dot plots corresponding to the end-point of the experiments. Mock-transfected cultures served to set the thresholds for background fluorescence (negative control). d Gene targeting in cells containing donor DNA resistant and susceptible to RGN nicking. Amplicons diagnostic for homology-directed gene targeting involving EGFP-encoding and mTurquoise2-encoding donor templates are indicated. HPRT1 provided for an internal control target sequence
