Generation of CRISPR/Cas9 knockout cell lines.

A549 MAVS KO and PKR KO cells were generated by using the CRISPR/Cas9 technology. Briefly, single guide RNA (sgRNA) sequences targeting the exon regions of PKR (5′-CAGGACCTCCACATGATAGG-3′) and MAVS (5′-CCGACCGGAAGTTCCAGGAG-3′) were selected using CHOPCHOP (1), a web tool for genome editing. A nontargeting sgRNA (5′-CTAAGGTTAAGTCGCCCTCG-3′) was used as a control. Annealed oligonucleotides were cloned into pLenti CRISPR v2 ccdB (2, 3) via a single-step Golden Gate cloning approach. In order to transduce A549 cells with the respective gRNA sequence, lentiviral VSV-G pseudotyped particles were produced in the cells. Therefore, 293T cells were transfected with plasmid DNA encoding the human immune deficiency virus (HIV) Gag and Pol proteins, VSV-G protein, and the respective pLenti CRISPR construct. Lentiviruses were harvested 48 h posttransfection, and A549 cells were transduced with the lentiviruses for 8 h. At 48 h postransduction, cells were subjected to puromycin selection at a concentration of 2 μg/ml. The batch knockout cell population was further diluted by limiting dilutions and seeded at ∼0.5 to 2 cells/well in 96-well plates, and single-cell clones were expanded for 3 weeks. Clones were characterized by immunoblotting.