CRISPR-Cas9 Plasmid Design for Innate Immunity

For CRISPR–Cas9 plasmids, single-guide RNA (sgRNA) targeting TBK1, IRF3, cyclic GMP-AMP synthase (cGAS), AP-1σ1 and STING were designed using the web tool CRISPOR^{47 (link)}. sgRNAs targeting TBK1, IRF3, cGAS and AP-1σ1 were cloned in a pSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid (Addgene, 62988), whereas sgRNAs targeting STING were cloned in a pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene, 48138). Both plasmids were gifts from F. Zhang^{48 (link)}. The pEF-Bos-based STING truncations (1–341 and 1–317) and mutations (E360A, LI364/365AA, ELI360/364/365AAA, LR374/375ΑΑ, L364A, L364F and I365A) were obtained by site-directed mutagenesis. pEF-Bos-human TBK1-Flag-His was a gift from S. Cerboni. TBK1(S172A) was generated by single-amino-acid mutation. pCDNA3-HA γ-adaptin 1(AP1G1) (Addgene, 10712) was purchased from Addgene. pCDNA3-HA-AP1S1 was generated by inserting the coding sequences of AP1S1 flanked by 5’ BamHI and 3’ XhoI sites into the pCDNA3 vector. AP1G1(R15E), AP1S1(I103S) and AP1S1(V88D) were obtained by single-amino-acid mutagenesis. The primers used for plasmid constructions and sgRNA sequences are provided in Supplementary Table 1. Plasmids for NF-κB-Luc (Promega, E8491) were purchased from Promega and those for pIFNβ–GLuc were previously described^{8 (link)}. All constructs were confirmed by DNA sequencing.