Inhibition Of Hepatitis B Virus Replication By CRISPR/Cas9 System And The Risk Evaluation Of Induced Viral DNA Integration

The hepatitis B virus(HBV)infection is one of the serious global public health problems.Currently,interferons and nucleotide analogs are the commonly used drugs for anti-HBV treatment,which can effectively reduce the amount of HBV in the serum of patients.However,it is prone to cause drug resistance mutations,and it is hard to completely eliminate the covalently closed circular DNA(cccDNA)of virus in the host cells.Recently,a new genome editing tool,CRISPR/Cas9 can be exploited to perform precise gene editing in a wide range of species,making it possible to fundamentally eradicate ccc DNA efficiently and conveniently.In this study,a specific CRISPR/Cas9 system targeting HBV genome was designed and constructed to study its effects on suppression of HBV DNA replication and antigen expression in HepG2.2.15 cells.Simultaneously,through whole genome sequencing,the risk of induced viral integration was analyzed after cleavage of cccDNA by the CRISPR/Cas9system.Firstly,we designed and screened the sequence targeting HBV genome,then constructed the recombinant sgRNA expression vector p U6-X3sgRNA and pU6-17sgRNA.After sequencing and identification,the recombinant sgRNA expression vector and hCas9 plasmid were co-transfected into HepG2.2.15 cells.After 48h,the hepatitis B virus surface antigen and HBV DNA,which existed in the cell serum,were detected by electrochemiluminescence immunoassay and quantitative real-time PCR respectively,also statistically analyzed and compared with the blank control group.Meanwhile,in order to investigate the risk of induced viral integration following by the cleavage cccDNA by CRISPR/Cas9 system,this study simulated its cleavage process and designed the green fluorescence protein(GFP)/sgRNA reporter system at first as follows:the circular control group(pCircular GFP+phCas9+psgRNA-control),the linear control group(p Linear GFP+phCas9+psgRNA-control)and circular experimental group(pCircular GFP+pCas9+psgRNA-GFP)plasmids were transfected into HepG2 cells respectively.After selected by puromycin,the expression of green fluorescence protein of each group were detected by inverted fluorescence microscopy and flow cytometry.Then the CRISPR/Cas9system that effectively inhibited the viral replication and expression were selected to transfected into HepG2.2.15 cells for 48h.Then the HBV DNA and HBsAg titer were detected.After the 15 days of continuous culture,we conducted the whole genome sequencing and bioinformatic analysis of viral DNA integration.The results showed that the titer of virus in the CRISPR/Cas9 system mediated by U6-X3sgRNA was(2.80±0.95)?10~4 IU/mL,significantly lower than that of the blank control group(6.17±0.17)?10~4 IU/m L,and the titer of HBsAg(1.25±0.01)IU/mL was also significantly lower than that of the blank control group(2.34±0.06)IU/mL,indicating that the CRISPR/Cas9 system mediated by U6-X3sgRNA could effectively inhibit the virus replication and expression.The result of green fluorescence protein reporter system conducted for evaluating for integration risk,showed that the expression level of GFP in the cells of circular experimental group and linear control group was significantly higher than that of experimental control group,implying that CRISPR/Cas9 could promote the integration of GFP after cleavage.However,in the CRISPR/Cas9 cleavage of ccc DNA experiment,compared with the control group,the whole genome sequencing and virus integration analysis revealed no more integration sites existed in the HepG2.2.15 cells after processed by CRISPR/Cas9 system;furthermore,PCR amplification also did not detect more fragments integration.Therefore,the hCas9-X3sgRNA system designed and constructed in this study could cleave specific cccDNA and effectively inhibit HBV DNA replication and antigen expression.And no risk of genomic random integration was found after the cccDNA fragments were cleaved by CRISPR/Cas9 system.