CRISPR activation and interference screens decode stimulation responses in primary human T cells [Bulk RNA-seq]

Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin 2 and interferon gamma production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization, revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA-seq enabled deep molecular characterization of screen hits, revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. Together, these screens reveal genes that reprogram critical immune cell functions, which could inform the design of immunotherapies. Overall design: RNA-seq of CD4+ human T cells after CRISPR activation of FOXQ1 in restimulated and resting conditions

活化T细胞的细胞因子产生调控过程，可在自身免疫性疾病、免疫缺陷病及癌症中发生紊乱。系统性发现刺激依赖性细胞因子调控因子，需同时开展功能丧失（loss-of-function）与功能获得（gain-of-function）研究，但此类研究在原代人细胞中极具挑战性。本研究报道在原代人T细胞中开展全基因组CRISPR激活（CRISPRa）与CRISPR干扰（CRISPRi）筛选，以鉴定调控白细胞介素2与干扰素γ产生的基因网络。阵列化CRISPRa验证了关键候选靶点，并支持多重分泌组特征分析，揭示了重塑的细胞因子应答模式。将CRISPRa筛选与单细胞RNA测序（single-cell RNA-seq）相结合，可对筛选得到的候选靶点开展深度分子表征，阐明基因扰动如何调控T细胞活化，并催生以独特细胞因子表达谱为特征的细胞状态。综上，本项筛选揭示了可重编程关键免疫细胞功能的基因，可为免疫治疗的设计提供理论依据。实验整体设计：在再次活化与静息状态下，对经CRISPR激活叉头框Q1（FOXQ1）的CD4+人T细胞开展RNA测序。