Rapid and efficient generation of antigen‐specific isogenic T cells from cryopreserved blood samples

Clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9)‐mediated gene editing has been leveraged for the modification of human and mouse T cells. However, limited experience is available on the application of CRISPR/Cas9 electroporation in cryopreserved T cells collected during clinical trials. To address this, we aimed to optimize a CRISPR/Cas9‐mediated gene editing protocol compatible with peripheral blood mononuclear cells (PBMCs) samples routinely produced during clinical trials. PBMCs from healthy donors were used to generate knockout T‐cell models for interferon‐γ, Cbl proto‐oncogene B (CBLB), Fas cell surface death receptor (Fas) and T‐cell receptor (TCRαβ) genes. The effect of CRISPR/Cas9‐mediated gene editing on T cells was evaluated using apoptosis assays, cytokine bead arrays and ex vivo and in vitro stimulation assays. Our results demonstrate that CRISPR/Cas9‐mediated gene editing of ex vivo T cells is efficient and does not overtly affect T‐cell viability. Cytokine release and T‐cell proliferation were not affected in gene‐edited T cells. Interestingly, memory T cells were more susceptible to CRISPR/Cas9 gene editing than naïve T cells. Ex vivo and in vitro stimulation with antigens resulted in equivalent antigen‐specific T‐cell responses in gene‐edited and untouched control cells, making CRISPR/Cas9‐mediated gene editing compatible with clinical antigen‐specific T‐cell activation and expansion assays. Here, we report an optimized protocol for rapid, viable and highly efficient genetic modification in ex vivo human antigen‐specific T cells, for subsequent functional evaluation and/or expansion. Our platform extends CRISPR/Cas9‐mediated gene editing for use in gold‐standard clinically used immune‐monitoring pipelines and serves as a starting point for development of analogous approaches, such as those including transcriptional activators and/or epigenetic modifiers.