A simple and efficient workflow for generation of knock‐in mutations in Jurkat T cells using CRISPR/Cas9

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.

Using CRISPR to enhance T cell effector function for therapeutic applications

“…The CRISPR/Cas9 toolbox can be used for silencing or knocking out genes [48] , [95] , [96] , knocking in genes [44] , [90] , and for the induced activation of genes in T cells [97] , [98] , [99] , [100] , [101] . Through these different approaches, various research questions have been investigated in T cells.…”

“…Several tools are available for the genetic modification of T cells, such as transcription activator-like effector nucleases, or TALENs, zinc finger nucleases, or ZFNs, and transposon-mediated genome editing [39] , [40] , [41] , [42] , [43] . Recently, also the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated (Cas) system was successfully applied in T cell cell-lines [44] , but has also been used to genetically modify primary murine and human T cells [45] , [46] , [47] , [48] , [49] . Specifically, CRISPR/Cas9 has been employed to directly enhance T cell effector function [48] , [50] , [51] , [52] , [53] , [54] , [55] , [56] , disable inhibitory receptor expression [45] , [57] , [66] , [67] , [68] , [58] , [59] , [60] , [61] , [62] , [63] , [64] , [65] , or to redirect T cell antigen specificity by targeting TCRs or CARs to the endogenous TCR-α chain ( TRAC ) locus [28] , [29] , [30] , [69] , [70] , [71] .…”

Adaptor proteins: Flexible and dynamic modulators of immune cell signalling

“…This may allow testing of single mutations of conserved amino acids, which may pinpoint their importance or even function. Such mutations can be further tested using genome editing technology such as CRISPR/Cas9, 211 which mediates deciphering roles of adaptor proteins in signalling pathways in relevant cell lines.…”

CRISPR/Cas9 high-throughput screening in cancer research

“…Firstly, adeno-associated vectors (AAV) are constructed and different sgRNA sequences are inserted in to establish the screening library [34]. As shown in the figure 2, SgRNA library was packaged with AAV and transferred to cas9 expression cell line with low virus infection complex number to ensure that each cell entered only one virus, to achieve functional screening for different sgRNA corresponding genes [35].…”