RT Journal Article SR Electronic T1 Efficient CRISPR/Cas9 gene ablation in uncultured naïve mouse T cells for in vivo studies JF bioRxiv FD Cold Spring Harbor Laboratory SP 730812 DO 10.1101/730812 A1 Simone Nüssing A1 Imran G. House A1 Conor J. Kearney A1 Stephin J. Vervoort A1 Paul A. Beavis A1 Jane Oliaro A1 Ricky W. Johnstone A1 Joseph A. Trapani A1 Ian A. Parish YR 2019 UL http://biorxiv.org/content/early/2019/08/09/730812.abstract AB 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.CRISPRclustered, regularly interspaced short palindromic repeats;Cas9CRISPR associated protein 9;Ctrlnon-targeting control sgRNA;sgRNAsingle guide RNA;LCMV-Cl13Lymphocytic choriomeningitis virus Clone 13 strain;LM-OVAovalbumin-transgenic Listeria monocytogenes