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Cell. 2018 Dec 13;175(7):1958-1971.e15. doi: 10.1016/j.cell.2018.10.024. Epub 2018 Nov 15.

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

Author information

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
UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, 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; 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.
#
Contributed equally

Abstract

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.

KEYWORDS:

CRISPR; T cell activation; T cell proliferation; genome-wide pooled screens; immunotherapy; primary human T cells; single-cell RNA-seq

PMID:
30449619
DOI:
10.1016/j.cell.2018.10.024

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