The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo

Abstract

Persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) under current antiviral therapy is a major barrier to eradication of chronic hepatitis B (CHB). Curing CHB will require novel strategies for specific disruption of cccDNA. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a newly developed tool for site-specific cleavage of DNA targets directed by a synthetic guide RNA (gRNA) base-paired to the target DNA sequence. To examine whether this system can cleave HBV genomes, we designed eight gRNAs against HBV of genotype A. With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector. Among eight screened gRNAs, two effective ones were identified. Interestingly, one gRNA targeting the conserved HBV sequence acted against different genotypes. Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.

A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo

“…Several previous studies have explored the potential of the CRISPR/Cas9 system to target HBV cccDNA in mice by hydrodynamic injection of plasmid DNA [7][8][9][10][11]; however, therapeutically relevant in vivo delivery methods are still lacking [12]. To systematically identify potential sgRNAs targeting HBV DNA, we used the CRISPR DESIGN tool (http:// crispr.mit.edu) to design potential sgRNAs.…”

Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells

“…4a-j). Meanwhile, the CRISPR/Cas9 system has been applied to target other infective viruses, such as papillomavirus, type I herpes simplex virus and hepatitis B virus by other groups 26,29,30 . These results demonstrated the generality of the CRISPR/Cas9 system for application as either a viral genome editing tool or anti-viral treatment.…”

Genome-Editing Technologies: Principles and Applications

“…This technical advance could help to overcome certain regulatory hurdles associated with the use of transgenic crops. Finally, targeted nucleases have also been used to inactivate pathogenic genes to prevent viral (Lin et al 2014) or parasitic (Ghorbal et al 2014) infection, as well as to introduce knockin-specific factors capable of imparting pathogen resistance . Intriguingly, targeted nucleases could also serve as conduits for curbing mosquito-or insect-borne diseases through a technique known as gene drive (Burt 2003;Sinkins and Gould 2006), which harnesses genome editing to facilitate the introduction of a specific gene or mutation that can then confer a particular phenotype into a host and also be transmitted to its progeny (Windbichler et al 2011).…”