Abstract
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.
Highlights
Immunity to infection depends on adequate intracellular signalling in many different immune cell types. Elucidation of these signalling pathways and the function of single signalling proteins have often been performed in cell lines and rodents using a variety of protein-specific inhibitors, knock-down assays and knockout techniques to target the protein of interest
To explore the function of specific subdomains, cDNA encoding recombinant proteins with domain-specific mutations can be forcibly expressed in both primary cells and in cell lines
Such exogenous expression of cDNA may not be regulated by cellular processes and the resulting recombinant protein might compete with the endogenous protein
Summary
Immunity to infection depends on adequate intracellular signalling in many different immune cell types Elucidation of these signalling pathways and the function of single signalling proteins have often been performed in cell lines and rodents using a variety of protein-specific inhibitors, knock-down assays and knockout techniques to target the protein of interest. These techniques have proven effective, many signalling molecules contain multiple functional domains. To explore the function of specific subdomains, cDNA encoding recombinant proteins with domain-specific mutations can be forcibly expressed in both primary cells and in cell lines. Such exogenous expression of cDNA may not be regulated by cellular processes and the resulting recombinant protein might compete with the endogenous protein
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