Efficient CRISPR/Cas9 Gene Editing in Uncultured Naive Mouse T Cells for In Vivo Studies

The Journal of Immunology : Official Journal of the American Association of Immunologists
Simone NüssingIan A Parish

Abstract

CRISPR/Cas9 technologies have revolutionized our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene editing strategies in T cells require in vitro stimulation or culture that can both preclude the study of unmanipulated naive T cells and alter subsequent differentiation. In this study, we demonstrate highly efficient gene editing within uncultured primary naive 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 knockout cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. Finally, we demonstrate that targeted mutations can be introduced into naive CD8+ T cells using CRISPR-based homology-directed repair. This protocol thus expands CRISPR-based gene editing approaches beyond models of robust T cell activation to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumor models.

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Citations

Sep 16, 2020·Nature Reviews. Drug Discovery·Simon J HoggRicky W Johnstone
Sep 6, 2020·The Journal of Immunology : Official Journal of the American Association of Immunologists·Samarchith P KurupJohn T Harty
Oct 31, 2020·The Journal of Experimental Medicine·Daiki MoriBernard Malissen
Nov 22, 2020·Methods and Protocols·Mateusz Kotowski, Sumana Sharma
May 30, 2021·Nature Communications·Lauren GiuffridaPaul A Beavis
Jun 4, 2021·The AAPS Journal·Amanda Catalina Ramirez-Phillips, Dexi Liu
Jun 9, 2021·Nature Immunology·Samuel A RoseUNKNOWN Immunological Genome Consortium
Aug 10, 2021·Frontiers in Immunology·Snigdha MajumderFriederike Berberich-Siebelt

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