The potential and challenges of CRISPR-Cas in eradication of hepatitis B virus covalently closed circular DNA

Current antiviral therapy fails to cure chronic hepatitis B virus (HBV) infection, primarily because of the persistence of covalently closed circular DNA (cccDNA). Although nucleos(t)ide analogues (NAs) can inhibit the reverse transcriptase of HBV and suppress its replication to levels below the detection limit, viremia often rebounds after cessation of therapy. Nuclear cccDNA serves as the HBV replicative template and exhibits extraordinary stability, and is not affected by NAs. Therefore, curing chronic hepatitis B (CHB) requires novel therapy for purging cccDNA from patients. The CRISPR/Cas9 system is a newly developed programmable genome-editing tool and allows for sequence-specific cleavage of DNA. Compared to other genome-editing tools, the CRIPSR/Cas9 system is advantageous for its simplicity and flexibility of design. Theoretically, Cas9 can be redirected to specifically cleave any desired genome sequences simply by designing guide RNAs with about 20 nucleotides that match the particular sequences of genomes with downstream protospacer adjacent motifs. Recently, it has been demonstrated that the CRIPSR/Cas9 system can specifically destruct HBV genomes in vitro and in vivo. Although promising, the CRISPR/Cas9 system faces several challenges that need to be overcome for the clinical application, namely, off-target cleavage and the in vivo delivery efficiency. Cutting integrated HBV genomes by CRISPR/Cas9 also raises serious concern because this has the risk of genome instability. In summary, the CRISPR/Cas9 system bears the potential for curing CHB as long as several challenging issues can be successfully solved.