[Construction of macrophage RAW 264.7 cells with gsdmd gene knockout by CRISPR/Cas9 system].

OBJECTIVE To construct a cell model of gsdmd gene knockout in macrophage RAW 264.7 cells using CRISPR/Cas9 system. METHODS Four specific single guide RNAs (sgRNAs) targeting gsdmd were designed to construct pGL3-sgRNA recombinant plasmids, which were identified by PCR amplification and sequencing.Cas9 and the recombinant plasmids were transfected into RAW 264.7 cells in two steps, and the cellular expression of cas9 was detected with real-time quantitative PCR (qPCR).The positive cell clones with gsdmd gene knockout were screened using puromycin and verified by sequencing and Western blotting.Annexin Ⅴ/PI staining and LDH release assay were performed in gsdmd-/-RAW 264.7 cells after being co-cultured with Salmonella Typhimurium. RESULTS qPCR results showed that cas9 gene was stably expressed in RAW 264.7-Cas9 cells (P< 0.01).PCR and sequencing results demonstrated successful construction of the recombinant plasmid pGL3-sgRNA. The results of PCR, sequencing and Western blotting all confirmed that gsdmd-/-RAW 264.7 cells were successfully constructed. Annexin Ⅴ/PI staining and LDH release assay showed that gsdmd gene knockout significantly inhibited macrophage death caused by S.Typhimurium infection (P < 0.01). CONCLUSIONS gsdmd-/-RAW 264.7 cells provide a cell model for studying the mechanisms underlying GSDMD-mediated macrophage death.