CRISPR-Cas9 Knockouts of Key Signaling Proteins

RIG-I, STING, STAT2, and p65 CRISPR-Cas9 KO cell lines were performed by transfection with Lipofectamine CRISPRMAX of TrueGuide Synthetic CRISPR gRNA and TrueCut Cas9 Protein v2 according to the manufacturer’s instructions (Thermo Fisher Scientific). CRISPR gene-editing efficiency was verified using GeneArt Genomic Cleavage Detection kit (A24372; Thermo Fisher Scientific). MAVS KO cell lines were performed by transfection of the pSpCas9(BB)-2A-Puro (PX459) plasmid containing MAVS gRNAs with FuGENE 6 Transfection Reagent (E2691; Promega). Sixteen hours after transfection, cells were selected with puromycin (1 μg/ml) for 2 days. IRF3 KO cell lines were performed by transfection of the pU6-(BbsI)-CBh-Cas9-T2A-mCherry plasmid containing IRF3 gRNAs with FuGENE 6 Transfection Reagent (E2691; Promega). Twenty-four hours after transfection, mCherry-expressing cells were single cell-sorted into 96-well plates. MCF10A IRF3 KO and MCF10A p53 KO cell lines was created as previously described^{66 ,84 (link)}. For every target, three or more independent clones were generated. gRNA sequences used in this study are listed in Supplementary Table 2.