Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function

Abstract

SUMMARYHuman T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, 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 validated hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell 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 response to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.

Paralog dependency indirectly affects the robustness of human cells

“…CS therefore reflects the relative deleteriousness of LOF on cell proliferation ( Fig EV1): A lower CS value indicates more deleteriousness and vice versa. These datasets are (i) CS1 from four cell lines (Wang et al, 2015), (ii) CS2 from 450 cell lines (Meyers et al, 2017;DepMap, 2018), (iii) CS2.1 from 450 cell lines (DepMap, 2018), and (iv) CS3 from 1 primary cell line (Shifrut et al, 2018; see Dataset EV2 for cell-line information, Dataset EV3 for gene-wise CS values). All the CS values capture the essentiality of the genes which, in the case of cancer cell lines, are found to be largely independent of the role of the genes in cancerogenesis ( Fig EV1).…”

“…The extent of depletion is measured as a CRISPR score (CS, see Materials and Methods). CS values across cell lines from three biologically independent datasets-CS1 (Wang et al, 2015), CS2/CS2.1 (Meyers et al, 2017;DepMap, 2018), and CS3 (Shifrut et al, 2018) are shown. Genes that are not in the paralog datasets but that were not identified as singletons in the stringent identification of singletons are denoted as "unclassified".…”

“…CRISPR score dataset CS3 (Shifrut et al, 2018) CS values for the CS3 dataset were obtained from a genome-wide CRISPR-Cas9 screening experiment in primary T cells (Shifrut et al, 2018). This dataset serves as an independent reference to the cancer or immortalized cell lines used in the other datasets.…”

“…These genes would have fitness effects that are closer to that of single copy genes (singletons) than that of typical duplicates. Here, we examine these predictions by re-analyzing a set of well-curated pairs of human paralogous genes (Singh et al, 2015;Lan & Pritchard, 2016) and recent large-scale genome-wide CRISPR-Cas9 screens in which the effect of gene LOF on cell proliferation was examined in more than 450 cancer cell lines (Wang et al, 2015;DepMap, 2018) and a primary cell line (Shifrut et al, 2018). The meta-analysis of the effect of gene LOF on cell proliferation, mRNA expression from 374 cell lines, protein expression from 49 cell lines and protein-protein interactions (Table EV1) revealed patterns which strongly support our hypothesis that paralogs that assemble are less protective, but through factors other than heteromerization itself.…”

A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

“…Use of the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR‐associated protein 9 (Cas9) system has revolutionized genome editing both in human (Shifrut et al, ) and microorganisms (Chen, Zhang, Yeo, Bae, & Ji, ; Vyas, Barrasa, & Fink, ). Briefly, the system requires two core components: a functional Cas9 protein to generate DNA double‐strand breaks (DSBs) and a single guide RNA (sgRNA) to guide the Cas9 endonuclease to any targeted locus in the chromosome with an appropriate protospacer adjacent motif (PAM).…”