CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X

Abstract

Current antiviral therapies cannot cure hepatitis B virus (HBV) infection; successful HBV eradication would require inactivation of the viral genome, which primarily persists in host cells as episomal covalently closed circular DNA (cccDNA) and, to a lesser extent, as chromosomally integrated sequences. However, novel designer enzymes, such as the CRISPR/Cas9 RNA-guided nuclease system, provide technologies for developing advanced therapy strategies that could directly attack the HBV genome. For therapeutic application in humans, such designer nucleases should recognize various HBV genotypes and cause minimal off-target effects. Here, we identified cross-genotype conserved HBV sequences in the S and X region of the HBV genome that were targeted for specific and effective cleavage by a Cas9 nickase. This approach disrupted not only episomal cccDNA and chromosomally integrated HBV target sites in reporter cell lines, but also HBV replication in chronically and de novo infected hepatoma cell lines. Our data demonstrate the feasibility of using the CRISPR/Cas9 nickase system for novel therapy strategies aiming to cure HBV infection.

Evolution in Our Understanding of Hepatitis B Virus Virology and Immunology

“…Several recent reports have used this strategy and designed guides for HBV sequences that were effective in disrupting episomal cccDNA and chromosomally integrated HBV, consequently disrupting HBV replication. [62][63][64][65] Despite promising results and convincing rationale, safety concerns arising from genome editing of host DNA independent of the HBV genome remain, because current CRISPR/ Cas9 approaches are known for low frequency but significant off-target cleavage and recombination events.…”

The implication of CRISPR/Cas9 genome editing technology in combating human oncoviruses

“…Since off‐targeting cleavage is the major drawback of CRISPR/Cas9, Cong et al and Ran et al described that the off‐target cleavage could be avoided by using nickase mutants of Cas9. Later on, Karimova et al also used Cas9 nickase to target the conserved sequences present in X and S HBV genome, which destroyed the cccDNA of HBV together with chromosomally integrated HBV genome inhibiting the viral replication. Strikingly, experiments of Li et al recently proved that by using CRISPR/Cas9 it is possible to completely excise 3175bp‐integrated HBV DNA along with the distortion of cccDNA of HBV.…”

Genome editing and the next generation of antiviral therapy

“…CRISPR/Cas9 is able to inhibit HBV replication and target cccDNA in a number of in vitro models (Karimova et al 2015; Kennedy et al 2014a; Liu et al 2015a; Ramanan et al 2015; Seeger and Sohn 2014; Wang et al 2015). It has also shown the ability to suppress HBV antigen expression from plasmids in a mouse hydrodynamic injection model of HBV that lacks cccDNA (Lin et al 2014; Zhen et al 2015).…”