CRISPR-Cas9 Knockout of DPP4 in Huh7 Cells for MERS-CoV Infection Study

LentiCRISPR-DPP4 plasmid was constructed by the subcloning of DPP4 gRNA sequence (5- CTTAGAATACAACTACGTGA-3) into the LentiCRISPR (Addgene 52961). Lentivirus expressing DPP4 gRNA and Cas9 was produced by the transfection of LentiCRISPR-DPP4, psPAX2 (Addgene 12260) and pCMV-VSV-G (Addgene 8454) into 293FT cells (Life Technologies). Huh7 cells were transduced with LentiCRISPR-DPP4 virus and further selected with puromycin dihydrochloride (Life Technologies) at 3.5 µg/ml in 10% FBS MEM. Cells were observed every 24 h for cell death. The transduced cells were subcultured to T75 culture flask for expansion when all the control cells were dead while the transduced cells stayed alive. hDPP4 expression in selected knockout cells was determined by western blot and IF staining. Purified CRISPR-Cas9 hDPP4-knockout Huh7 cells were subject to infection with Ty-BatCoV HKU4 SM3A. Infectivity assay was performed as previously described at MOI 1^{33 (link)}. Both supernatants and cell lysates were collected at 0, 1, 2, 3, 5 and 7 dpi. Viral load in supernatants was determined using RT-qPCR.