Rapid and efficient generation of antigen-specific isogenic T cells from cryopreserved blood samples
ObjectivesCRISPR/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 e.g. clinical trials. MethodsPBMCs from healthy donors were used to generate knockout T cell models for interferon-{gamma} (IFN{gamma}), Cbl Proto-Oncogene B (CBLB), Fas cell surface death receptor (Fas) and T cell receptor (TCR{beta}b) 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. ResultsOur 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 naive 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. ConclusionHere, 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.