Abstract
Genetic manipulation of human primary T cells is a valuable technique for basic research in immunology to explore gene function and for discovering novel clinical applications. Electroporation is the most feasible non-viral material delivery system for manipulating human T cells given its time- and cost-effectiveness. However, efficient delivery requires electroporation settings to be optimized for different electroporation devices, cellular states, and materials to be delivered. In this study, we used electroporation to either induce exogenous gene expression in human primary T cells by plasmids or in vitro transcribed (IVT) mRNA and also target endogenous genes by Cas9 ribonucleoproteins (RNPs). We characterized the electroporation conditions both for activated and unstimulated T cells. Although naive cells are non-dividing and therefore their genetic manipulation is harder compared to activated T cells, we developed the technical ability to manipulate both naive and memory cells within the unstimulated T cell population by IVT mRNA and Cas9 RNP electroporation with more than 95% and 80% efficiency, respectively, and by plasmids with more than 50% efficiency. Here, we outline the best practices for achieving highly-efficient and non-viral genetic manipulation in primary T cells without causing significant cytotoxicity to the cells. Because there is increasing evidence for “less-differentiated” T cells to have a better anti-tumor activity for immunotherapy, manipulating naive T cells with high efficiency is also of high importance to clinical applications. Furthermore, manipulation of naive T cells without the need for activation is important for studying the biology of these cells.
