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.
Cell. 2018 Dec 13;175(7):1958-1971.e15. doi: 10.1016/j.cell.2018.10.024. Epub 2018 Nov 15.
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.
人类 T 细胞是免疫和癌症免疫疗法的核心效应器。基于 CRISPR 的 T 细胞功能研究可以为药物开发确定新的优先靶点，并改进基因重编程细胞治疗的设计。然而，在原代人细胞中进行大规模的 CRISPR 筛选具有挑战性。我们开发了一种新方法，即 Cas9 蛋白电穿孔的单指导 RNA（sgRNA）慢病毒感染（SLICE），以鉴定原代人 T 细胞刺激反应的调节因子。全基因组功能丧失筛选鉴定了关键的 T 细胞受体信号传导成分和基因，这些基因在刺激后负调控增殖。对单个候选基因的靶向消融鉴定了增强癌细胞杀伤的命中靶点和扰动。SLICE 与单细胞 RNA 测序（RNA-seq）相结合，揭示了受关键遗传扰动改变的特征性刺激反应基因程序。SLICE 全基因组筛选也适用于鉴定免疫抑制的介质，揭示控制腺苷信号反应的基因。SLICE 平台可用于在原代细胞中进行无偏发现和功能基因靶点的鉴定。
