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Riggan, Luke; Hildreth, Andrew D.; Rolot, Marion; Wong, Yung-Yu; Satyadi, William; Sun, Ryan; Huerta, Christopher; O’Sullivan, Timothy E.
Cell reports, 05/2020, Letnik: 31, Številka: 7Journal Article
CRISPR genome engineering has become a powerful tool to functionally investigate the complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity, the utility of current approaches is restricted by poor knockout efficiencies or limited specificity for mature cell lineages in vivo. Here, we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of mature primary mouse innate lymphocyte cells (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore, we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naive natural killer (NK) cells and bone-marrow-derived conventional dendritic cell precursors (cDCPs). This resource will enhance target gene discovery and offer a specific and simplified approach to gene editing in the mouse innate immune system. Display omitted •Optimized electroporation of cRNP complexes in primary mature innate immune cells•High knockout efficiency of signaling adaptors and transcription factors•cRNP-mediated knockout of Stat4 in primary NK cells during MCMV infection•cRNP knockout reveals MyD88 is required for cDC1-dependent control of MCMV infection Riggan et al. optimize electroporation conditions for high-efficiency cRNP-mediated gene deletion in primary mature mouse innate immune cells and utilize this approach to elucidate gene function of NK cells and cDC1s using two in vivo adoptive transfer models during MCMV infection.
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