Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication

Figure 3 Eleven dual gRNAs could efficiently suppress the production of hepatitis B virus surface antigen or hepatitis B virus e antigen. The plasmid pBB4.5-HBV1.2 was co-transfected with gRNA-1 and -13 (A), gRNA-8 and -12 (B), or gRNA-1 and -13 (C) expression vectors to HuH-7 cells, alone or in combination. HBsAg and HBeAg levels in cell culture supernatant were measured at 72 h post transfection using a time-resolved fluoroimmunoassay; D: The plasmid pBB4.5-HBV1.2 (0.5 μg) was co-transfected with different combinations of two gRNAs expression vectors (each 0.75 μg) to HuH-7 cells. HBsAg level in culture supernatant was measured at 72 h post transfection using a time-resolved fluoroimmunoassay; E: HBeAg level in culture supernatant was measured using time-resolved fluoroimmunoassay as above; F: The cytotoxicity of dual gRNAs was examined using an MTT assay. Data are shown as mean ± SE of 3 independent experiments. All P-values are from Student’s t-test. HBV: Hepatitis B virus; HBsAg: Hepatitis B virus surface antigen; HBeAg: Hepatitis B virus e antigen; MTT: Methyl thiazolyl tetrazolium.