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.

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A.N., and A.S. designed experiments and analyzed results. P.B. and V.S. performed immunoblotting, IP, and flow cytometry experiments. P.B. generated mutated cell lines. G.A. performed NFAT translocation assay. H.C. performed Lck-SH2 domain PD. H.K. performed molecular cloning of all necessary constructs. T.A.N. analyzed mass spectrometry data. P.B. and A.