Transcriptional adaptation after deletion of <i>Cdc42</i> in primary T cells

Cdc42 is a Rho family GTPase known for its central role in cell polarity and cytoskeletal regulation. To understand the role of Cdc42 in polarised secretion from cytotoxic T lymphocytes (CTLs) we used CRISPR/Cas9 gene deletion. Although Cdc42-deleted CTLs initially showed reduced cytotoxicity for up to 2 days after CRISPR-mediated deletion, full secretion and cytotoxicity was rapidly restored and even enhanced while CDC42 protein remained absent. In contrast, chemical inhibition of CDC42 using CASIN consistently decreased secretion in wild-type cells, but had no impact on Cdc42-deleted CTLs, confirming the specificity of this inhibitor. Comparative proteomics and transcriptomics of CTLs after Cdc42 deletion revealed transcriptional changes that could support improved T cell function, including compensation via other Rho GTPases. Targeting the promoter region of Cdc42 did not trigger transcriptional adaptation, consistent with a nonsense-mediated decay mechanism of genetic compensation. Our work highlights the importance of taking orthogonal approaches to study protein function and reveals the remarkable robustness of primary T cells to adapt to loss of an essential gene.