Application of CRISPR/Cas9 Technology to HBV

Abstract

More than 240 million people around the world are chronically infected with hepatitis B virus (HBV). Nucleos(t)ide analogs and interferon are the only two families of drugs to treat HBV currently. However, none of these anti-virals directly target the stable nuclear covalently closed circular DNA (cccDNA), which acts as a transcription template for viral mRNA and pre-genomic RNA synthesis and secures virus persistence. Thus, the fact that only a small number of patients treated achieve sustained viral response (SVR) or cure, highlights the need for new therapies against HBV. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system can specifically target the conserved regions of the HBV genome. This results in robust viral suppression and provides a promising tool for eradicating the virus. In this review, we discuss the function and application of the CRISPR/Cas9 system as a novel therapy for HBV.

Keywords: CRISPR/Cas9; HBV; antiviral; cccDNA.

Figure 1

The life cycle of hepatitis B virus (HBV) with therapeutic targets. HBV binds to surface receptors and enters the hepatocyte. Viral particles migrate to the cell nucleus, where the HBV genomes are converted to a covalently closed circular (cccDNA) that serves as a template for viral transcription. The translation of viral mRNA in the cytoplasm results in the production of the core (C), polymerase (P), surface (S) and hepatitis B virus X (HBx) proteins. Next, genomic viral RNA is packaged into the progeny viral capsids. The core particle can either be encapsulated and secreted from the hepatocyte or be reimported into the nucleus for transformation to cccDNA. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-directed disruption of the HBV life cycle can target (A) or (B) the entry receptor sodium taurocholate co-transporting polypeptide (NTCP) which is necessary for the HBV life cycle.

Figure 2

Schematic of CRISPR/Cas9 mediated-genome editing. (A) The sgRNA guides the Cas9 protein to cut specific DNA sequence by recognizing the protospacer-adjacent motif (PAM) sequence and a complementary target sequence; (B) The breaks induced by Cas9 are repaired either through homology-directed repair (HDR) or non-homologous end joining (NHEJ), results in precise gene editing with insertions or deletions (indels), respectively. NGG: PAM sequence.

Figure 3

Illustration of HBV targeting strategy and possible mechanism of CRISPR/Cas9 in suppression of HBV. C: core; P: polymerase; S: surface; X: HBx.