A CRISPR-Based Toolbox for Studying T Cell Signal Transduction

Abstract

CRISPR/Cas9 system is a powerful technology to perform genome editing in a variety of cell types. To facilitate the application of Cas9 in mapping T cell signaling pathways, we generated a toolbox for large-scale genetic screens in human Jurkat T cells. The toolbox has three different Jurkat cell lines expressing distinct Cas9 variants, including wild-type Cas9, dCas9-KRAB, and sunCas9. We demonstrated that the toolbox allows us to rapidly disrupt endogenous gene expression at the DNA level and to efficiently repress or activate gene expression at the transcriptional level. The toolbox, in combination with multiple currently existing genome-wide sgRNA libraries, will be useful to systematically investigate T cell signal transduction using both loss-of-function and gain-of-function genetic screens.

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 sgRNA^Control or the sgRNA^B2M. 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 sgRNA^B2M 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).

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 sgRNA^Control or sgRNA^CD28. 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).

Figure 3

A Jurkat T cell line with sunCas9 system. (a) In the CRISPRa system, dCas9 fused to SunTag is able to recruit multiple copies of VP64 and can activate gene transcription. (b-c) sgRNA expressions in JS19 cells can substantially upregulate their targeting gene expressions. JS19 cells were transduced with virus expressing the sgRNA^Control (blue histograms), the sgRNA^CXCR4 (red histogram in (b)), or the sgRNA^PDGFRB (red histograms in (c)). Cells were grown for more than a week and then analyzed for CXCR4 expression (b) and PDGFRB expression (c) in the transduced cells. The grey histograms represent the negative control (unstained sample). Data are shown in histograms and are representative of three independent experiments.