Generation of KO cell lines by CRISPR/Cas9

CRISPR/Cas9 was used to KO STING, TBC1D9, and TBK1. CRISPR guide (g)RNAs were designed to target an exon common to all splicing variants of the gene of interest (5′-GAGAGTGTGCTCTGGTGGC-3′ for STING, 5′-AACCCGGAGGAGGTGTTGC-3′ for TBC1D9, and 5′-GAGCACTTCTAATCATCTG-3′ for TBK1). HeLa cells were transfected with the vector hCAS9 (Addgene #41815) and a gRNA-hyg vector containing the CRISPR target sequence. Untransfected cells were removed by selection on plates containing 300 μg mL⁻¹ hygromycin B (Nacalai Tesque) and 750 μg mL⁻¹ geneticin (G418; Nacalai Tesque). Single colonies were expanded, and depletion of the target gene was confirmed by immunoblot. As a secondary screen for some KO lines, genomic DNA was isolated, and target regions were amplified by PCR and sequenced to confirm the presence of the desired frameshift insertions and deletions.