Eradication Of HBV Infection Using CRISPR-Cas9

Chronic hepatitis B(CHB)caused by hepatitis B virus(HBV)is a major public health problem worldwide,with 1 million deaths a year as a result of hepatocellular carcinoma,cirrhosis of the liver or liver failure caused by HBV infection.Although,the wide application of vaccine prevented the spread of HBV to a largely extent,the treatment of CHB was still in delimma.Nucleoside/nucleotide analogs(NAs)are regarded as effective intervention,while,NAs could only emporarily inhibite the replication of HBV and rebound viremia often occurs after the cessation of antiviral treatment.Current antiviral treatments have little or no ability to eliminate replicative HBV templates,including covalently closed circular DNA(ccc DNA)and integrated HBV DNA.Given the shortcomings of current therapeutic options,treatment of HBV infection represents a global challenge and highlights the need to approach CHB in a fundamentally different way.Recently,a new genome-editing tool,CRISPR-Cas9,was developed based on the bacterial immune system’s clustered regularly interspaced short palindromic repeats(CRISPRs).CRISPR-Cas9 is predicated to be a promising genome-editing tool with broad applications,which makes it possible to eradicate the HBV ccc DNA and integrated HBV DNA.In this study,using CRISPR-Cas9,we disrupted the replicative HBV templates to efficiently suppress markers of viral replication both in HBV stable cell line and HBV hydrodynamics mouse model.Also,we completely excised the full-length integrated HBV DNA and HBV ccc DNA in a stable HBV cell line.In addition we removed the full length of integrated HBV DNA,which means that we achieved a “sterile” eradication of HBV infection in this stable HBV cell line.To assess the clearance of integrated subgenomic HBV DNA fragments and the risk of the comprehensive use of the CRISPR-Cas9 system,whole-genome sequencing(WGS)was performed on the cell lines before and after HBV excised.Unexpectedly,we found millions of mutations in HBV stable cell line changed after HBV infection and excision.1.Suppressing HBV replication and expression by CRISPR-Cas9(1)Screening and optimization of an effective HBV-specific CRISPR/Cas9 system.The HBV virion contains many overlapping open reading frames(ORFs)and there are many variations in HBV genome and different forms of HBV DNA in host cell.To selecte the most efficient g RNA target,we compared the HBV genome sequences of different subtypes and identified an effective g RNA/Cas9 system that could disrupt HBV DNA to efficiently suppress markers of viral replication with minimal off-target effects from 8 candidate g RNAs.Also,we confirmed the disruption of HBV DNA and evalued the cleavage efficiency of g RNA using deep sequencing.(2)Specific inactivation of HBV replication by CRISPR/Cas9 both in vitro and in vivoTo evalue the inactivation effect of HBV replication by gRNA/Cas9 in vitro and in vivo,we used HBV stable cell line and HBV hydrodynamics mouse as model.In this part of study,we efficiently suppressed viral replication in HBV stable cell line,we also demostrated that the reduction of HBV replication(95.37 ± 0.64% reduction of HBV ccc DNA)was caused by the mutation of S4 site through three S4 region-mutated monoclonal cells.Besides,the g RNA-S4 system could also reduce serum surface-antigen levels by 99.92 ± 0.04% and lowered serum HBV DNA level below the negative threshold in the HBV hydrodynamics mouse model.(3)Process analysis of disrupting HBV DNA by CRISPR-Cas9In above results,the rate of indel at S4 site was much lower than the percentage declines in HBV antigens.It possibly because that circular HBV DNA such as HBV ccc DNA had been effectively cleaved into a high percentage of linear DNAs which is not repaired but destroyed.As three forms of the envelope protein HBs Ag and the viral polymerase were encoded by the overlapping ORF where designed S4 target right located,targeting one site on HBV genome could make the overall reduction of HBV.2.Removal of the integrated HBV DNA using CRISPR-Cas9The integration of HBV DNA into the host genome can cause alterations of the host genome,leading to changes in genes associated with cell proliferation,differentiation,and survival,which is also an important factor in hepatocarcinogenesis.Besides HBV ccc DNA,the functional full-length integrated HBV DNA is also a replication root in an HBV-transgenic cell model.Thus,only to disrupt the HBV ccc DNA and remove the full length of integrated HBV DNA,can we completely eradicate the HBV infection in the stable HBV cell line.As the life cycle of HBV in stable HBV cell line is similar to natural HBV infection and there are also many forms of HBV DNA such as HBV ccc DNA and HBV DNA inside the viral nucleocapsid in this cell model,eradication of integrated HBV DNA in this cell model could simulate the real condition of clearance the integrated HBV DNA fragments in clinical CHB patients.(1)Establishment of a new method to amplify the full-length integrated HBV DNAIn HBV-infected cells,the existence of many different forms of episomal HBV DNA and multiple integrated HBV sites in different chromosomes made it cumbersome to specifically amplify the full-length of integrated HBV DNA site in a stable HBV cell line using Alu-PCR or next-generation sequencing(NGS).Using a specific primer derived from the foreign promoter of the full-length replication-competent integrated HBV DNA,we established a new method to amplify and sequencing the full-length of integrated HBV DNA in a stale HBV cell line.(2)First excised the full-length integrated HBV genome and eradicate HBV infection in stable HBV cell lineWe completely excised a full-length 3,175-bp integrated HBV DNA fragment and disrupted HBV ccc DNA in a stable HBV cell line.In HBV-excised cell line,the HBV ccc DNA inside cells,supernatant HBV DNA,HBs Ag,and HBe Ag remained below the negative critical values for more than 10 months.It is the first time that the HBV infection has been fully eradicated in a stable HBV cell line.3.WGS Analysis of host genome before and after HBV excised(1)The majority of integrated subgenomic HBV DNA fragments were missing in HBV excised cell lineTo assess the clearance of integrated subgenomic HBV DNA fragments and the risk of the comprehensive use of the CRISPR-Cas9 system,whole-genome sequencing was performed on the cell lines before and after HBV excised.Compared with A64 cell line before HBV excised,the HBV integrated sites has reduced 97.7% in HBV excised cell line 69-7,which showed that the integrated subgenomic HBV DNA fragements were missing after HBV infection had been totally eradicated.(2)Millions of mutations in the HBV-excised cells were recovered to the state before HBV infectionAfter comparing the mutations especially the SNPs in HBV stable cell line Hep G2.A64,HBV excised cell line 69-7 and their initial cell line Hep G2,we found that there were 2,284,202 SNP sites variated after HBV infection,in which 2,093,238 SNP sites recovered to the state before HBV infection.It showed that using CRISPR-Cas9 to disrupt the HBV DNA and eradicate the infection could recover millions of mutations caused by HBV infection rather than induce the extra mutations.(3)Evaluate the off target effect of gRNA-69 and exclude other possibilities for those recovered mutationsTo make a further investigation of those mutations recovery after HBV excised,we predicted the off target sites of g RNA-69 and analysis those sites in the sequencing results.The results showed that g RNA-69 did not have any significant off-target effect.Next,after analysis of HBV integration sites,we proved that HBV excised cell line 69-7 was not the Hep G2 contaminants in stable HBV cell line Hep G2.A64.Also,using a specific residual sequence of g RNA-69/Cas9 cleavage,we confirmed that the HBV-excised cell line was established by g RNA-69/Cas9 and was not exist in a very small amount as contaminants in stable HBV cell line Hep G2.A64 before g RNA-69 treatment.(4)Putting forward a hypothesis for the mutations recovery and HBV integrated sites missing after HBV excisedAfter analysis the variant types of those recovered mutations after HBV excised,we found that the most variant types were from heterozygous SNPs to homozygous SNPs or directly recovered from heterozygous SNP.Thus,we inferred that those recoverd SNPs were repaired by the DNA repaired system with the homologous chromosomes.Then,we analyzed the expression of RAD51,a critical gene in DNA homologous recombination pathway,in cell lines before and after HBV excised.The results showed that the expression of RAD51 was suppressed after HBV infection and reactived after the infection been eradicated.In conculsion,it is the first time to eradicate HBV infection from host cell using CRISPR-Cas9 system.We also analyzed the off-target effect,cleavage efficiency and action duration in this process.Unexpectedly,using the whole genome sequencing,we found that the majority of the integrated subgenomic HBV DNA fragments were missing and millions of host mutations were recovered to the state before HBV infection.The results in this study showed the promising usage of CRISPR-Cas9 for the cure of HBV infections and HBV-related diseases.It also provides new insights into the mechanism underlying HBV DNA fragment integration and the genetic architecture of viral-host genetic interactions.