CRISPR/Cas9 technologies have revolutionised our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene deletion strategies in T cells require in vitro stimulation or culture that can both preclude studies of gene function within unmanipulated naïve T cells and can alter subsequent differentiation. Here we demonstrate highly efficient gene deletion within uncultured primary naïve murine CD8 + T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knock-out cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naïve state, suggesting that gene deletion occurs independent of T cell activation. This protocol thus expands CRISPR-based probing of gene function beyond models of robust T cell activation, to encompass both naïve T cell homeostasis and models of weak activation, such as tolerance and tumour models.