How to clear HBV cccDNA: CRISPR/Cas9 might be promising ABSTRACT BACKGROUND: Chronic hepatitis B infected with Hepatitis B virus remains a major health concern worldwide. Despite standard interferon-α and nucleotide analogues have been shown to reduce the deterioration of liver disease among chronic

BACKGROUND: Chronic hepatitis B infected with Hepatitis B virus remains a major health concern worldwide. Despite standard interferon-α and nucleotide analogues have been shown to reduce the deterioration of liver disease among chronic hepatitis B patients, covalently closed circular DNA was still difficult to eradicate. METHODS: A literature search of Pubmed and Web of science was performed with the following key words: ‘CRISPR’, ‘CRISPR/Cas9’, ‘hepatitis B’, ‘HBV’, ‘chronic hepatitis B’ and ‘HBV cccDNA’. The information about CRISPR/Cas9 for the treatment of HBV cccDNA or hepatitis B was reviewed. RESULTS: CRISPR/Cas9 could treat hepatitis B through suppressing or clearing HBV cccDNA with different gRNAs. CONCLUSION: With the emergence of CRISPR/Cas9 (the RNA-guided clustered regulatory interspaced short palindromic repeats, CRISPR) editing technology, clearance of hepatitis B virus and better prevention of liver carcinoma seemed to be possible.


Introduction
Hepatitis B virus (HBV) infection is still a major public health problem affecting 248 million people worldwide [1].It has caused 780,000 deaths annually due to hepatitis B related diseases such as cirrhosis and hepatocellular carcinoma (HCC) [2,3].Up till now, there are seven agents extensively used for the treatment of chronic hepatitis B (CHB) including standard interferon-α (IFN-α) or pegylated interferon-α (PEG-IFN-α) and five nucleotide/ nucleoside analogues (NA).Although both IFN and NA could reduce the progression of liver disease in CHB patients, there are no satisfied needs of existing CHB treatment to clear the covalently closed circular DNA (cccDNA) and better prevention of HCC.
Due to HBV cccDNA, the transcription template for viral mRNA and pregenomic RNA synthesis and secures virus persistence, eradication of HBV infection still poses a great challenge.Fortunately, several strategies to cure HBV are always in research , including strategies to inhibit the nucleocapsid assembly, HBV RNA, HBV entry receptor, host immune responses, HBV cccDNA and new polymerase inhibitors.As to the strategy to eradicate HBV cccDNA, there are three most common nuclease-initiated genome editing tools: the zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the RNA-guided clustered regulatory interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein endonucleases [26,27].In the past few decades, ZFNs and TALENs have attracted extensive attention because of their advantages.
Studies found that ZFNs can be used to inhibit viral transcription and replication of duck HBV through blocking the transcription of cccDNA [28] , while TALENs could target the conserved regions of HBV DNA and reduce the cccDNA levels [29].
However, the time-consuming and costly engineering of the target gene-specific fusion proteins are prominent disadvantages.Therefore, a novel easy genome editing tool entered the spotlight: the CRISPR/Cas9 technology [30].This article reviews and discusses the current researches of the novel therapeutic technology-CRISPR/Cas9-to target HBV cccDNA from basic medical perspective.

Cell culture models
With the advantages of being more convenient and faster than in vivo experiments, cell culture are very practical to perform cellular researches in a well-controlled experimental setting.As with the emergence of CRISPR/cas9 cells [13,47,[49][50][51][52][53][54][55][56][57][58].Among them, Huh-7 and HepG2 cells with 1.2 or 1.3 copies of HBV genome were more often used for their practices and conveniences.
HepG2/NTCP cells were also used for its capability of infecting 1.05 copies of HBV genome in contrast with parental HepG2 cells without NTCP transfection [16].
However, Zhu et al. [59] showed that there were no significant differences of anti-HBV productions by pCas9-2 constructs exists in between Huh-7 and HepG2 hepatoma cells.After transfection with different Cas9/gRNA plasmid vectors, the cells were used for analyzing the effect of CRISPR/Cas9 system on the disruption or suppression of hepatitis B virus.

Animal models
As cultured cells may respond differently to HBV infection and other stimuli than cells in the living organ [59], the capability to express various hepatocyte-specific genes might be lost.Hence, it should consider data discrepancies obtained in vitro and in vivo, and verify the observations from cell cultures in animal models.
According to Maura Dandri [60], there are three types of mouse models of HBV replication up to now.These are transgenic mice, vector-medicated transduction of HBV in mice and hydrodynamic injection of replication competent HBV [61] (figure 1).As mice cannot be infected with HBV but with the advantages of the best characterized and most convenient small laboratory animal, genetically modified mice were generated for expressing either viral proteins or the full HBV genome with great efforts of researchers.In 1995, the first HBV-replicating transgenic mice were developed, and the murine hepatocytes could produce infectious HBV virions morphologically indistinguishable from human-derived virions [62].
Since the HBV genome is integrated in the host genome and cccDNA is not build in murine hepatocytes, Maura Dandri thought that viral clearance cannot be achieved.Meanwhie, the mechanisms of cccDNA synthesis and strategies aiming at destabilizing the cccDNA cannot be investigated [63].Therefore, the targeting of CRISPR/Cas9 nucleases to cccDNA in transgenic mice was all tested combined with hydrodynamic tail-vein injection technology.For instance, Li H [56] and his colleagues delivered gRNA-S4 system to the liver of adult HBV-Tg mice using

Remaining Issues and Potential Solutions
Although the advancing CRISPR/Cas9 gene editing technology is now at an interesting stage to eradicate the persistent HBV genome, there are still some remaining issues needing to be addressed.Of them, cytotoxic seemed to have been solved in most studies.
First, the efficiency of CRISPR/Cas9 system is low needing to be improved.In some studies [13,50], serum HBsAg level was reduced but persisted following the CRISPR/Cas9 therapy, potentially leading to rebound viremia and repopulation of HBV in the liver after the cessation of the antiviral therapy.To overcome the hurdle, an efficient delivery system with a high vector, such as adenoviral [67,68] or adeno-associated virus (AAV) vectors [68,69], to target cell ratio and achieve continuous and prolonged treatment.Hence, it may be possible for the use of immediate, prolonged treatment and the combination of several treatments.
The second issue is the off-target effect, which is pertinent concerns for all genome-editing strategies and may cause non-specific gene modification events.To utilize the CISPR/Cas9 system for cure of HBV infection, it is best to eliminate this off-target activity.The issue may be solved by designing truncated gRNAs [70], paired nickase gRNAs [71] and gDNAs [72].Of them, the gDNA guided NAgo system, which has lower off-target effect and does not require any protospacer-adjacent motif (PAM) sequence, has been confirmed suitable for genome editing in human cells.
The third issue is the fraction of cccDNA possibly existing in minichromosomes.
Consequently, minichromosomes of cccDNA can still be targeted and cleaved remains unclear, needing further investigation.

Conclusions
Although hepatitis B could be suppressed with drugs, cccDNA could not be fully eradicated.With great efforts of researchers, many new strategies have been used to treat HBV.Of them, CRISPR/Cas9 has been demonstrated to target HBV cccDNA with different gRNAs.Meanwhile, experimental models in vitro and in vivo have been established almost successfully.As to cell culture studies, Huh-7 and HepG2 cells with 1.2 or 1.3 copies of HBV genome were more often used than HepG2/NTCP cells with the capability of infecting 1.05 copies of HBV genome.While involving the animal models, three types of mouse models of HBV replication including transgenic mice, vector-medicated transduction of HBV in mice and hydrodynamic injection of replication competent HBV were gradually improved for CRISPR/Cas9 treatment of HBV.Undoubtedly, there was still some remaining problems related needing to be solved.Hence, further studies should be conducted to perfect the CRISPR/Cas9 system for HBV therapy.
hydrodynamic tail-vein injection to confirm the effect.Zhu W et al.[55] injected pCas9 constructs into the M-Tg HBV mouse model of HBV by hydrodynamics and found decreased HBsAg of sera and liver HBcAg.Zhen S and his colleagues also used HBV-Tg mice after delivering Cas9 and gRNA-S1+X3 plasmid by hydrodynamics.They showed reduced HBsAg secreted in mouse serum and almost no HBsAg-positive cells in the liver tissue of CRISPR/Cas9-S1+X3-treated mice[50].Although the HBV-Tg mouse lineage might be produced in different background (C57/BL or BALB/c), the transgene present in these mice consists 1.3 copies of HBV genome and expresses high levels of HBsAg in their serum and has detectable levels of HBV DNA in their serum[61,64,65].Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 10 October 2017 doi:10.20944/preprints201710.0065.v1Apart from the transgenic mouse model, alternative mouse models have been developed.They relied on the transfer of HBV genomes into mice by using viral vector mediated transduction or hydrodynamic injection of HBV genome.However, viral vector mediated transduction combined with the hydrodynamic injection was more used in the application of CRISPR/Cas9 to treat CHB.At present, there were four researches that used both the models [13, 49, 51, 52].In the research that the CRISPR/Cas9 system facilitates clearance of intrahepatic HBV templates in vivo, pAAV/HBV 1.2 and gRNA/Cas9 dual expression vector were injected into the tail veins of HBV persistent mouse model with C57BL/6 background.In another study that targeting HBV cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication, precccDNA plasmid, pCre plasmid and pX330/pCas9 plasmid were injected into the tail vein of female Balb/c mice.Vyas Ramanan [52] and his colleagues used the hydrodynamic delivery technique to inject 1.3xHBV plasmid and CRISPR expressing plasmid to NRG mice for studying the effect of CRISPR/Cas9 on the suppressing HBV.Similarly, Liu et al. conducted hydrodynamics-based transfection in mice, that is, injecting pHBV1.3 and pSV-β-gal, pX330-U6-HBV gRNA plasmids or pX 330-U6-GFPgRNA plasmid into the tail vein of BALB/c mice [49].Through all the above mouse models and different gRNA, they demonstrated the inhibition or cleavage effect of CRISPR/Cas9 to target HBV cccDNA and other parameters of viral gene expression and replication.