Shifrut and Carnevale et al. developed a high-throughput CRISPR screening platform that works directly on ex vivo human T cells by combining sgRNA lentiviral infection with Cas9 protein electroporation (SLICE). Genome-wide specific disruption of target genes led to the identification of positive and negative regulators of proliferation following TCR stimulation and regulators of suppression following adenosine signaling. Knockdown of some negative regulator genes identified by SLICE enhanced target cell killing by T cells in vitro. Combining SLICE with RNAseq revealed how each gene manipulation can distinctly shape the cell state.
Human 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, 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.
Author Info: (1) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San
Author Info: (1) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA. (2) UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA. (3) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA. (4) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA. (5) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA. (6) UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA. (7) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA. (8) UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA. (9) UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA. (10) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA. Electronic address: alexander.marson@ucsf.edu.
