AIM: To screen and investigate the effective g RNAs against hepatitis B virus(HBV) of genotypes A-D.METHODS: A total of 15 g RNAs against HBV of genotypes A-D were designed. Eleven combinations of two above g RNAs(dual-g RNAs) covering the regulatory region of HBV were chosen. The efficiency of each g RNA and 11 dual-g RNAs on the suppression of HBV(genotypes A-D) replication was examined by the measurement of HBV surface antigen(HBs Ag) or e antigen(HBe Ag) in the culture supernatant. The destruction of HBV-expressing vector was examined in Hu H7 cells co-transfected with dual-g RNAs and HBVexpressing vector using polymerase chain reaction(PCR) and sequencing method, and the destruction of ccc DNAwas examined in Hep AD38 cells using KCl precipitation, plasmid-safe ATP-dependent DNase(PSAD) digestion, rolling circle amplification and quantitative PCR combined method. The cytotoxicity of these g RNAs was assessed by a mitochondrial tetrazolium assay.RESULTS: All of g RNAs could significantly reduce HBs Ag or HBe Ag production in the culture supernatant, which was dependent on the region in which g RNA against. All of dual g RNAs could efficiently suppress HBs Ag and/or HBe Ag production for HBV of genotypes A-D, and the efficacy of dual g RNAs in suppressing HBs Ag and/or HBe Ag production was significantly increased when compared to the single g RNA used alone. Furthermore, by PCR direct sequencing we confirmed that these dual g RNAs could specifically destroy HBV expressing template by removing the fragment between the cleavage sites of the two used g RNAs. Most importantly, g RNA-5 and g RNA-12 combination not only could efficiently suppressing HBs Ag and/or HBe Ag production, but also destroy the ccc DNA reservoirs in Hep AD38 cells.CONCLUSION: These results suggested that CRISPR/Cas9 system could efficiently destroy HBV expressing templates(genotypes A-D) without apparent cytotoxicity. It may be a potential approach for eradication of persistent HBV ccc DNA in chronic HBV infection patients. AIM: To screen and investigate the effective g RNAs against hepatitis B virus (HBV) of genotypes AD. METHODS: A total of 15 g RNAs against HBV of genotypes AD were designed. Eleven combinations of two above g RNAs covering the regulatory region of HBV were chosen. The efficiency of each g RNA and 11 dual-g RNAs on the suppression of HBV (genotypes AD) replication was examined by the measurement of HBV surface antigen (HBs Ag) or e antigen ) in the culture supernatant. The destruction of HBV-expressing vector was examined in Hu H7 cells co-transfected with dual-g RNAs and HBV expressing vector using polymerase chain reaction (PCR) and sequencing method, and the destruction of ccc DNA was examined in Hep The cytotoxicity of these g RNAs was was assessed by a mitochondrial tetrazolium assay .RESULTS: All of g RNAs could have been found to have significant activity in AD38 cells using KCl precipitation, plasmid-safe ATP-dependent DNase (PSAD) digestion, rolling circle amplification and quantitative PCR combined method. antly reduce HBs Ag or HBe Ag production in the culture supernatant, which was dependent on the region in which g RNA against. All of the dual g RNAs could efficiently suppress HBs Ag and / or HBe Ag production for HBV of genotypes AD, and the efficacy of dual g RNAs in suppressing HBs Ag and / or HBe Ag production was significantly increased when compared to the single g RNAs used alone. Furthermore, by PCR direct sequencing we confirmed that these dual g RNAs could specifically destroy HBV expressing template by removing the fragment Most importantly, g RNA-5 and g RNA-12 combination only did not suppress HBs Ag and / or HBe Ag production, but also destroy the ccc DNA reservoirs in Hep AD38 cells. CONCLUSION: These results suggest that CRISPR / Cas9 system could efficiently destroy HBV expressing templates (genotypes AD) without apparent cytotoxicity. It may be a potential approach for eradication of persistent HBV ccc DNA in cchronic HBV infection patients.