Flow cytometry data - Computationally Engineered CRISPR-SpyCas9 High-Fidelity Variants with Improved Specificity and Reduced Non-specific DNA Damage

EGFP-PEST stable cells were co-transfected with one of the sgRNA plasmids and one of the Cas plasmids, together with a reporter plasmid. Five days post transfection cells were trypsinized, incubated 5 min at 37 °C, and neutralized by 150ul FBS-enriched FACS buffer (PBS; 5% FBS; 25mM HEPES Biological industries (Israel) 03-025; 5mM EDTA, Biological industries (Israel) 01-862). The suspended cells were filtered using a mesh-capped plate (Merck MANMN4010) before flow cytometry analysis. Cells were analyzed by flow cytometry (CytoFLEX S, Beckman Coulter) to assess GFP-disruption rates. Decrease in GFP levels was assessed by measuring the GFP positive cells, based on GFP+ untreated cells, out of mCherry positive cells. Each plate contained its internal positive and negative controls for normalization.