T-cell-based cancer immunotherapies have emerged as a promising approach for cancer treatment, highlighting the importance of understanding the regulation of T-cell function. However, the molecular mechanisms underlying T-cell activation are not fully understood. The CRISPR/Cas9 system can serve as a robust method to systematically study signaling pathways. In this chapter, we describe details of using the CRISPR screen to identify regulators in TCR signaling, from the sgRNA library construction to genomic DNA sequencing. We also add some notes to further help readers performing the CRISPR screen. This approach can be readily adapted to study the activation of other immune cells, including B cells and dendritic cells.
gRNA library T-cell activation Lentivirus production and titer Lentiviral transduction Cell sorting Genomic DNA extraction
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The authors thank F. Wang, Z. Lin (ShanghaiTech University), and J. M. Shen (The Semiconductor Manufacturing International Corporation Private School) for their critical reading of the manuscript. H.W. is funded by National Natural Science Foundation of China Grant 31670919 as well as the 1,000-Youth Elite Program of China.
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