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(dua...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.展开更多
基金Supported by Natural Science Foundation of China,No.81471938the National S and T Major Project for Infectious Diseases,No.2013ZX10002-002 and No.2012ZX10002-005111 Project,No.B07001
文摘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.
