Targeting Hepatitis B Virus With CRISPR/Cas9

Abstract

Hepatitis B virus persistence in infected hepatocytes is due to the presence of covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs. Antiviral therapies with nucleoside analogues inhibit replication of HBV DNA in capsids present in the cytoplasm of infected cells, but do not reduce or destroy nuclear cccDNA. To investigate whether cccDNA derived from infectious HBV could be directly targeted for destruction, we used the CRISPR/Cas9 system in HepG2 cells expressing the HBV receptor sodium taurocholate cotransporting polypeptide (NTCP). We tested different HBV-specific guide RNAs and demonstrated that they could inhibit HBV infections up to eightfold. Inhibition was due to mutations and deletions in cccDNA similar to those observed with chromosomal DNA cleaved by Cas9 and repaired by nonhomologous end joining (NHEJ). Interferon alpha (IFN-α) did not have a measurable effect on the antiviral activity of the CRISPR/Cas9 system, suggesting that Cas9 and NHEJ activities are not affected by induction of an innate immune response with the cytokine. Taken together, our results demonstrated that Cas9 can be recruited to cccDNA, opening the possibility for the development of future antiviral strategies aimed at targeting cccDNA for endonucleolytic cleavage with small molecules.

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

“…In eukaryotes, the DSBs are more commonly repaired by the mechanism of error-prone non-homologous end joining (NHEJ), therefore generating sequence changes, for instance insertions and deletions (indels), around the DSBs . Owing to the simplicity of manipulation and versatility, the CRISPR/Cas9 system has been utilized as an attractive tool for various applications, such as genome-wide screening (Shalem et al, 2014;Zhou et al, 2014), gene repression and activation (Cheng et al, 2013;Doench et al, 2014;Gilbert et al, 2014), targeted fluorescence imaging (Tanenbaum et al, 2014) and novel approaches against pathogens including hepatitis B virus (Lin et al, 2014a;Seeger & Sohn, 2014), human papillomavirus (Kennedy et al, 2014), Epstein-Barr virus (Wang & Quake, 2014;Yuen et al, 2015), malaria (Wagner et al, 2014) and HIV-1 (Ebina et al, 2013;Hu et al, 2014;Ye et al, 2014).…”

How to Clear HBV cccDNA: CRISPR/Cas9 Might Be Promising

“…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].…”

A novel phenotypic assay of hepatitis B virus polymerase with extensive site-specific mutagenesis

“…The replication capability under 0 μmol/L LAM can reflect the "natural" activity, while that under 20 μmol/L of LAM reflects the "fitness" under drug pressure. Intracellular nucleocapsid-associated HBV DNA levels were measured by real-time PCR, and mitochondria DNA was used as reference (Seeger and Sohn, 2014). The F1927 and R2051 primers were used in real-time PCR (Supplementary Table S1), and the reaction system contained 10 μL FastStart Universal SYBR Green Master (Roche Diagnostics, Mannheim, Germany), 2 μL of DNA, 0.1 μL (10 μmol/L) of each primer, and 7.8 μL of H 2 O.…”