title: Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9.
abstract: The presence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and the permanent integration of HBV DNA into the host genome confers the risk of viral reactivation and hepatocellular carcinoma. Nucleoside/nucleotide analogs alone have little or no capacity to eliminate replicative HBV templates consisting of cccDNA or integrated HBV DNA. Recently, CRISPR/Cas9 technology has been widely applied as a promising genome-editing tool, and HBV-specific CRISPR-Cas9 systems were shown to effectively mediate HBV cccDNA disruption. However, the integrated HBV DNA fragments are considered as important pro-oncogenic properties and it serves as an important template for viral replication and expression in stable HBV cell line. In this study, we completely excised a full-length 3,175-bp integrated HBV DNA fragment and disrupted HBV cccDNA in a stable HBV cell line. In HBV-excised cell line, the HBV cccDNA inside cells, supernatant HBV DNA, HBsAg, and HBeAg remained below the negative critical values for more than 10 months. Besides, by whole genome sequencing, we analyzed off-target effects and excluded cell contamination. It is the first time that the HBV infection has been fully eradicated in a stable HBV cell line. These findings demonstrate that the CRISPR-Cas9 system is a potentially powerful tool capable of promoting a radical or "sterile" HBV cure.
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Li
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Chunyu
authors.initials: C
authors.lastname: Sheng
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Shan
authors.initials: S
authors.lastname: Wang
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Lang
authors.initials: L
authors.lastname: Yang
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liang
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology Beijing, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Liu
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Chaojie
authors.initials: C
authors.lastname: Yang
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Yang
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Leili
authors.initials: L
authors.lastname: Jia
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Xie
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Ligui
authors.initials: L
authors.lastname: Wang
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Rongzhang
authors.initials: R
authors.lastname: Hao
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Xinying
authors.initials: X
authors.lastname: Du
authors.affiliation: Research Centre for Liver Failure, Beijing 302nd Hospital Beijing, China.
authors.forename: Dongping
authors.initials: D
authors.lastname: Xu
authors.affiliation: Research Center for Translational Medicine, Cancer Stem Cell Institute, East Hospital, Tongji University School of MedicineShanghai, China; Gladcan Consulting CompanyBeijing, China.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Research and Development Department, Beijing Center for Physical and Chemical Analysis Beijing, China.
authors.forename: Mingzhen
authors.initials: M
authors.lastname: Li
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Yansong
authors.initials: Y
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology Beijing, China.
authors.forename: Yigang
authors.initials: Y
authors.lastname: Tong
authors.affiliation: Department of Surgery, University of Michigan Ann Arbor, MI, USA.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Li
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Shaofu
authors.initials: S
authors.lastname: Qiu
authors.affiliation: Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
authors.forename: Hongbin
authors.initials: H
authors.lastname: Song
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002460:Cell Line; D004270:DNA, Circular; D018390:Gene Targeting; D016679:Genome, Viral; D006509:Hepatitis B / Q000382:microbiology*; D006515:Hepatitis B virus / Q000502:physiology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016662:Virus Integration; D014779:Virus Replication
chemical_list: D004270:DNA, Circular
keywords: CRISPR-Cas9; HBV cccDNA; hepatitis B virus; integrated HBV DNA; whole genome sequencing