Research of Genome-wide CRISPR Screening in Primary Human T Cells among Key Regulators of Immune Function

Introduction

Cytotoxic T cells play a central role in immune-mediated control of cancer, infectious diseases, and autoimmunity. Genome-scale CRISPR screens in primary human T cells would enable comprehensive target discovery studies that could be rapidly translated into new immunotherapies with small molecules, biologics, and gene-engineered adoptive cell therapies. Here, a screening platform was developed that combines pooled lentiviral sgRNA delivery with Cas9 protein electroporation to identify regulators of stimulation responses in primary human T cells. Further combining pooled CRISPR delivery with single-cell transcriptome analysis of human T cells allows characterization of several gene-controlled cellular programs found to regulate T cell responses in a genome-wide screen. The series of studies using this screening platform provides a rich resource of gene pathways to target and tune human T cell responses, which is important in the search for promising preclinical candidates for next-generation cellular therapies.

Methods

Primary human T cells for all experiments were sourced from residuals from leukoreduction chambers after Trima Apheresis from de-identified blood donors following written informed consent or from fresh whole blood samples under a protocol approved by the UCSF Committee on Human Research.

Lentiviral transduction and Cas9 electroporation were performed in order to establish a high-throughput CRISPR screening platform. To test the constructed screening platform, the sgRNA library was transduced into T cells from healthy donors, and the cells were restimulated to systematically identify genes that were positively or negatively selected in the proliferating T cell population. Next, to confirm that the hits were actually dependent on TCR stimulation and to confirm the ability of high scoring genes to promote T cell activation and proliferation through array electroporation of individual Cas9 ribonucleoproteins (RNP), separate GW screens were performed to test the effect of these array knockouts on T cell proliferation after TCR stimulation using high throughput flow cytometry. In addition, SLICE was coupled to droplet-based single-cell transcriptome reads for pooled perturbed high-dimensional phenotypes in primary human T cells via the CROP-seq platform. Finally, the effect of target gene knockdown in an antigen-specific in vitro cancer cell killing system was tested by engineering human T cells.

Methods

Results

The investigators of this article developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.

Fig. 1 Framework for Unbiased Discovery of Regulators of Human T Cell Proliferation Using Pooled CRISPR Screens.

Summary

Exposure of transfected Ba/F3 cells to increasing concentrations of molecular targeted drugs will often result in the development of drug resistance. The use of ENU can facilitate and shorten the process of resistance induction. One of the first applications of Ba/F3 cells for identifying secondary resistance mutations was reported by Ercan et al who used ENU mutagenesis and identified an EGFR C797S mutation as a mechanism of osimertinib resistance. Furthermore, Katayama et al used Ba/F3 cells to identify secondary ROS1 mutations that could cause crizotinib or ceritinib resistance.

Reference:

Shifrut, E.; et al. Genome-wide CRISPR screens in primary human T cells reveal key regulators of immune function. Cell, 2018, 175(7): 1958-1971. e15.

* For research use only. Not intended for any clinical use.

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Research of Genome-wide CRISPR Screening in Primary Human T Cells among Key Regulators of Immune Function

Introduction

Methods

Results

Summary

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