Supplementary Figure 3: Fluorescence-activated cell sorting of SpCas9 library-infected human cells harboring on- and off- target reporters.
From: Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9
OVCAR8-ADR reporter cell lines that express RFP and GFP genes driven by UBC and CMV promoters, respectively, and a tandem U6 promoter-driven expression cassette of gRNA targeting the RFP site (RFPsg5 or RFPsg8) were either uninfected or infected with the SpCas9 library. RFPsg5-ON and RFPsg8-ON lines harbor sites that match completely with the gRNA sequence, while RFPsg5-OFF5-2 and RFPsg8-OFF5 lines contain synonymous mutations on the RFP and are mismatched to the gRNA. Cells were sorted under flow cytometry into bins each encompassing ~5% of the population with low RFP fluorescence. These experiments were repeated independently twice with similar results.
