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

Figure 5 Hepatitis B virus-specific gRNA could suppress the genotype D hepatitis B virus replication and destroy cccDNA. A: HepAD38 cells were seeded into a 6 well plate. Then, gRNA-5 and -12 expression vectors (each 2 μg) were co-transfected into HepAD38 cells. HBsAg and HBeAg levels in the culture supernatant of HepAD38 cells were measured at 72 h post transfection using a time-resolved fluoroimmunoassay; B: HBV DNA levels in the culture supernatant of HepAD38 cells were measured at 72 h post transfection using real-time quantitative PCR; C: HBV cccDNA levels in HepAD38 cells were measured using KCl precipitation, plasmid-safe ATP-dependent DNase (PSAD) digestion, rolling circle amplification and quantitative PCR combined method; D: PCR amplification was performed using the primers beyond the cleavage sites of dual gRNAs following KCl precipitation, PSAD digestion and rolling circle amplification. HBV: Hepatitis B virus; HBsAg: Hepatitis B virus surface antigen. The asterisk means the digested fragment of cccDNA.