Pcmv hcas9

A XAF1 knockout CAD5 cell line was established by CRISPR/Cas9 genome editing approach. Briefly, a guide RNA targeting exon 1 of the XAF1 gene (5′-AGG​CTG​ACT​TCC​AAG​TGT​GC-3′) was cloned into the pKLV-U6gRNA(BbsI)-PGKpuro2ABFP vector (Addgene 50946) to generate pKLV-XAF1 single guide RNA plasmid. CAD5 cells were cotransfected with pKLV-XAF1 single guide RNA and pCMV-hCas9 (Addgene 41815) in a molar ratio of 1:1 by Lipofectamine 2000 (11668-019; Invitrogen). Transfected cells were selected and enriched by puromycin (2 µg/ml), and single-cell clones were obtained by limited dilution. Cell clones were grown and expanded. To confirm gene editing of the XAF1 gene in cell clones, a region embracing exon 1 of the XAF1 gene was PCR-amplified using a forward primer (5′-GTC​ACA​GTC​ACG​GTA​GCA​CA-3′) and reverse primer (5′-GGG​ACC​AGT​CCC​TCC​ATA​CT-3′); the PCR product was cloned into pCR-Blunt II-TOPO vector (45-0245; Invitrogen) and sequenced to verify the frameshifted indels.