title: 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.
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Su-Ru
authors.initials: SR
authors.lastname: Lin
authors.affiliation: 1] Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan [2] Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan [3] Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Hung-Chih
authors.initials: HC
authors.lastname: Yang
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Yi-Ting
authors.initials: YT
authors.lastname: Kuo
authors.affiliation: 1] Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan [2] Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan [3] Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Chun-Jen
authors.initials: CJ
authors.lastname: Liu
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Ta-Yu
authors.initials: TY
authors.lastname: Yang
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Ku-Chun
authors.initials: KC
authors.lastname: Sung
authors.affiliation: 1] Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan [2] Department of Life Science, National Taiwan University College of Life Science, Taipei, Taiwan.
authors.forename: You-Yu
authors.initials: YY
authors.lastname: Lin
authors.affiliation: Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Hurng-Yi
authors.initials: HY
authors.lastname: Wang
authors.affiliation: Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Chih-Chiang
authors.initials: CC
authors.lastname: Wang
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Yueh-Chi
authors.initials: YC
authors.lastname: Shen
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Fang-Yi
authors.initials: FY
authors.lastname: Wu
authors.affiliation: 1] Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan [2] Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan [3] Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan [4] Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Jia-Horng
authors.initials: JH
authors.lastname: Kao
authors.affiliation: 1] Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan [2] Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan [3] Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan [4] Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Ding-Shinn
authors.initials: DS
authors.lastname: Chen
authors.affiliation: 1] Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan [2] Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan [3] Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan [4] Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Pei-Jer
authors.initials: PJ
authors.lastname: Chen