A CRISPR-Based Toolbox for Studying T Cell Signal Transduction

Figure 2

Engineering of A Jurkat T cell line for dCas9-KRAB mediated gene silencing. (a) In the CRISPRi system, dCas9 fused to KRAB domain can repress transcription of target gene. (b) sgRNA expression in JK28 cells can remarkably downregulate its targeting gene expression. Jurkat cells stably expressing dCas9-KRAB protein were transfected with constructs expressing or . Cells were grown for 6 days and then analyzed for CD28 expression in the GFP+ transfected cells. Data are shown in histogram and are representative of three independent experiments. (c) Transcription repression mediated by dCas9-KRAB is reversible. JKBulk and JK28 cells were transfected with sgRNAs as described in (b). The percentages of the cells with reduced expression of CD28 were assessed by FACS at different time points after transfection. The data summarize the results of three independent experiments (data represent mean value ± SD). (d) sgRNA expression is a limiting factor for CRISPRi function. The transfected cells were divided into four populations according to their GFP expression. The percentage of cells losing CD28 expression was quantified by flow cytometry 6 days following transfection. The data summarizes the results of three independent experiments (data represent mean value ± SD). (e) Silencing CD28 expression by CRISPRi interference IL-2 production in activated T cells. IL-2 assays were performed in the indicated conditions. The data summarize the results of three independent experiments (data represent mean ± SD).

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