*2.2. Generation of Knock-Out Cells*

To stably knock out RIG-I and MDA5, sgRNAs cloned into pX458-Ruby (Addgene 110164, deposited by Dr. Philip Hublitz) and described earlier [13] were used. To stably knock out IFNAR1, a sgRNA targeting *IFNAR1* exon 3 was selected based on the MIT algorithm (crispr.mit.edu) and cloned into pX458 (Addgene 48138, deposited by Dr. Feng Zhang). A549 and HEK293 cells were single-cell FACS sorted according to the co-expressed fluorescent protein (Ruby+ for cells transfected with the sgRNAs targeting RIG-I or MDA5, GFP+ for IFNAR1) 48 h post transfection. After 4 weeks, cells that had grown out to confluency were subjected to cell line characterization. We extracted genomic DNA and analyzed the target locus with a PCR screening protocol using primers up- and downstream of the sgRNA target sites. Primer sequences were: RIG-I (fwd: ttacattgtctcagactaagaggc, rev: gtgaagaatgggcacagtcggcc), MDA5 (fwd: cgtcattgtcaggcacagag, rev: agctctgccactgtttttcc) and IFNAR (fwd: gtgtatgctaaaatgttaatagg, rev: cctttgcgaaatggtgtaaatgag). Full knock-out was verified by submission of sequencing reads to TIDE (https://tide.nki.nl), an algorithm that decomposes sequencing data and allows determination of the spectrum of indels and their respective frequencies. Additionally, whole cell lysates were analyzed by western blot after stimulation with recombinant type I IFN (IFN-A/D, Sigma, 100 U/mL).
