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

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.

Mineralogical characterization of copper sulfide tailings using automated mineral liberation analysis: A case study of the Chambishi Copper Mine tailings

As ore grades constantly decline,more copper tailings,which still contain a considerable amount of unrecovered copper,are expected to be produced as a byproduct of froth flotation.This research reveals the occurrence mechanism of copper minerals in typical copper sulfide tailings using quantitative mineral liberation analysis(MLA)integrated with scanning electron microscopy–energy dispersive spectroscopy(SEM–EDS).A comprehensive mineralogical characterization was carried out,and the results showed that almost all copper minerals were highly disseminated within coarse gangue particles,except for 9.2wt%chalcopyrite that occurred in the 160–180μm size fraction.The predominant copper-bearing mineral was chalcopyrite,which was closely intergrown with orthoclase and muscovite rather than quartz.The flotation tailings sample still contained 3.28wt%liberated chalcopyrite and 3.13wt%liberated bornite because of their extremely fine granularity.The SEM–EDS analysis further demonstrated that copper minerals mainly occurred as fine dispersed and fully enclosed structures in gangue minerals.The information obtained from this research could offer useful references for recovering residual copper from flotation tailings.