A CRISPR-Based Toolbox for Studying T Cell Signal Transduction

Figure 1

A Jurkat T cell line optimized for WT-Cas9 mediated genome editing. (a) WT-Cas9 generates DNA double-strand breaks at the targeted genome locus, resulting in disruption of the target gene. (b) JX17 cells achieve high genome editing efficiency. Jurkat cells stably expressing WT-Cas9 protein were transfected with constructs expressing the or the . Cells were grown for 6 days and then analyzed for MHC I expression in the GFP+ transfected cells. Data are shown in histogram and are representative of four independent experiments. (c) Disruption of gene by WT-Cas9 is irreversible. Jurkat cells were transfected with sgRNAs as described in (b). The expression of MHC class I was assessed by FACS at different time points after transfection. The chart summarizes the results of three independent experiments (data represent the mean value ± SD). (d) Loss of MHC class I expression was restored by exogenous expression of B2M gene. JX17 cells were electroporated with as described in (b). MHC class I-negative JX17 cells were sorted and electroporated with either an empty vector (blue histogram) or a plasmid expressing B2M gene (red histogram). The expression of MHC class I was assessed by FACS 48 hours after electroporation. The grey histogram represents the negative control (unstained sample). (e) WT-Cas9 edits genome in an sgRNA dose-dependent manner. The transfected cells were divided into four populations according to their GFP expression. The percentage of cells losing MHC I expression was quantified by flow cytometry 6 days following transfection. The chart summarizes the results of three independent experiments (data represent mean value ± SD).

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