CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell–based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies.

CRISPR（成簇、规则间隔、短回文重复序列）/Cas9（CRISPR 相关蛋白 9）已成为在多个物种中产生基因敲除的首选工具。原代 T 细胞中高效基因编辑的能力不仅代表了研究基因功能的宝贵研究工具，而且还为基于 T 细胞的免疫疗法（例如下一代嵌合抗原受体 (CAR) T 细胞）带来了巨大前景。先前尝试应用 CRIPSR/Cas9 在原代 T 细胞中进行基因编辑，导致敲除效率高度可变，并且需要 T 细胞受体 (TCR) 刺激，从而在很大程度上排除了参与 T 细胞激活或分化的基因的研究。在这里，我们描述了一种在没有 TCR 刺激的情况下对原代小鼠和人类 T 细胞进行 Cas9/RNP 转染的优化方法，该方法导致群体水平上的靶基因表达几乎完全丧失，从而减轻了选择的需要。我们相信，这种方法将极大地扩展原代T细胞中靶基因发现和验证的可行性，并简化下一代免疫疗法的基因编辑过程。