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.

将EGFP-PEST稳定细胞株（EGFP-PEST stable cells）与单向导RNA（single-guide RNA，sgRNA）质粒、Cas质粒以及报告质粒共转染。转染后第5天，对细胞进行胰酶消化，于37℃条件下孵育5分钟，随后采用150 μL富含胎牛血清的荧光激活细胞分选（Fluorescence-Activated Cell Sorting，FACS）缓冲液进行中和，该缓冲液组分如下：磷酸盐缓冲液（PBS）、5%胎牛血清（Fetal Bovine Serum，FBS）、25 mM 4-羟乙基哌嗪乙磺酸（HEPES，以色列Biological industries公司，货号03-025）、5 mM 乙二胺四乙酸（EDTA，以色列Biological industries公司，货号01-862）。将重悬后的细胞通过带筛网滤板（Merck MANMN4010）过滤后，开展流式细胞术分析。使用CytoFLEX S流式细胞仪（贝克曼库尔特Beckman Coulter）检测细胞，以评估GFP敲除效率。以未处理的GFP阳性细胞为参照，通过统计mCherry阳性细胞中的GFP阳性细胞占比，以评估GFP水平的下降幅度。每块实验板均设置内部阳性对照与阴性对照，用于实验结果的归一化处理。