CRISPR/Cas9 is a powerful gene‐editing tool allowing for specific gene manipulation at targeted sites in the genome. Here, we used CRISPR/Cas9‐mediated gene editing to introduce single amino acid mutations into proteins involved in T cell receptor signalling pathways. Knock‐in mutations were introduced in Jurkat T cells by homologous directed repair using single‐stranded oligodeoxynucleotides. Specifically, we aimed to create targeted mutations at two loci within LCK, a constitutively expressed gene, and at three loci within SH2D2A, whose expression is induced upon T cell activation. Here, we present a simple workflow that can be applied by any laboratory equipped for cell culture work, utilizing basic flow cytometry, Western blotting and PCR techniques. Our data reveal that gene editing may be locus‐dependent and can vary between target sites, also within a gene. In our two targeted genes, on average 2% of the clones harboured homozygous mutations as assessed by allele‐specific PCR and subsequent sequencing. We highlight the importance of decreasing the clonal heterogeneity and developing robust screening methods to accurately select for correct knock‐in mutations. Our workflow may be employed in other immune cell lines and acts as a useful approach for decoding functional mechanisms of proteins of interest.

中文翻译：

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使用CRISPR / Cas9在Jurkat T细胞中产生敲入突变的简单高效的工作流程

CRISPR / Cas9是功能强大的基因编辑工具，允许在基因组中的目标位点进行特定的基因操作。在这里，我们使用CRISPR / Cas9介导的基因编辑将单个氨基酸突变引入涉及T细胞受体信号传导途径的蛋白质中。使用单链寡聚脱氧核苷酸通过同源定向修复将敲入突变引入Jurkat T细胞。具体来说，我们的目标是在组成性表达基因LCK的两个基因座和SH2D2A的三个基因座创建靶向突变，其表达在T细胞活化后被诱导。在这里，我们介绍了一个简单的工作流程，可以利用基本的流式细胞仪，Western印迹和PCR技术，由配备了细胞培养功能的任何实验室应用。我们的数据表明，基因编辑可能是基因座依赖的，并且在目标位点之间也可能在基因内变化。通过等位基因特异性PCR和随后的测序评估，在我们的两个靶向基因中，平均有2％的克隆具有纯合突变。我们强调了降低克隆异质性并开发可靠的筛选方法以准确选择正确的敲入突变的重要性。我们的工作流程可以在其他免疫细胞系中使用，并作为解码目标蛋白质功能机制的有用方法。