STING gene knockout pig alveolar macrophage cell model constructed by CRISPR/Cas9 technology and its effect on type Ⅰ interferon transcription

【Objective】To elucidate the mechanism of interferon gene stimulating factor(STING) in the anti-pathogenic microbial infection of pigs,so as to further provide a reference for the scientific prevention and control of viral diseases such as porcine transmissible gastroenteritis,epidemic diarrhea and porcine pseudorabies.【Method】High-scored targets were found in exons 4 and 8 of STING gene and corresponding sgRNA sequences were designed based on CRISPR/Cas9 technology. The annealed sgRNAs were linked with the enzyme digested LentiCRISPRV2 carrier with T4 DNA ligase to obtain LentiCRISPRV2-STING-sgRNA lentivirus carrier(STING-sgRNA);Different combinations of STING sgRNA lentivirus carriers,packaging plasmid psPAX2 and envelope plasmid pMD2.G were transfected into 293T cells to obtain lentivirus containing sgRNA and then transduced into 3D4/21 cells. Monoclonal cell lines were obtained by puromycin screening and limited dilution method. The knockout efficiencies of the STING gene were identified by PCR amplification,Sequencing and Western blotting;The effect of STING gene knockout on the expression of type Ⅰ interferon was verified by real-time fluorescent quantitative PCR.【Result】When 293T cells were transfected with different combinations of STING-sgRNA lentivirus carrier and HA-STING over expression vector,the editing effect of STING eukaryotic expression carrier could be detected in cells,and the combination of STING-sgRNA(1+5)lentivirus carrier showed the supreme editing efficiency. Thus,the STING-sgRNA(1+5)lentivirus carrier combined with the packaging plasmid psPAX2and the envelope plasmid p MD2.G were transfected 293T cells to package lentivirus,and then infected 3D4/21 cells with lentivirus. The results showed that a 3D4/21 cell line with a large deletion of the STING gene(4989 bp)was obtained.The STING protein was not observed by Western blotting,indicating that the STING gene knockout 3D4/21 cells(3D4/21-STING^-/-)were successfully constructed. The transcription level of IFN-β in 3D4/21-STING^-/-cells decreased significantly(P<0.05)compared with parental cells when stimulated by transfection of Haemophilusparasuis DNA.【Conclusion】By applying CRISPR/Cas9 technology,STING gene is successfully knock out in 3D4/21 cells,resulting in loss of function of STING gene;STING knockout leads to the transcription disorder of type Ⅰ interferon when cells are stimulated by DNA,which also suggests that STING gene may be a key factor in the anti-pathogenic microbial infection of pigs.