Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9

Frontiers in Cellular and Infection Microbiology

Liang, Yuan¹  Yang, Chaojie¹  Li, Hao¹  Liu, Hongbo¹  Sheng, Chunyu¹  Yang, Xiaoxia¹  Du, Xinying¹  Xie, Jing¹  Yang, Lang¹  Tong, Yigang¹  Li, Peng¹  Song, Hongbin¹  Hao, Rongzhang¹  Wang, Ligui¹  Jia, Leili¹  Wang, Shan¹  Qiu, Shaofu²  Li, Mingzhen³  Zhou, Jianjun⁴  Xu, Dongping⁵  Sun, Yansong⁶  Huang, Yong⁷  Li, Qiao⁷ 

[1] Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China;Department of Surgery, University of Michigan, Ann Arbor, MI, USA;Gladcan Consulting Company, Beijing, China;Research Center for Translational Medicine, Cancer Stem Cell Institute, East Hospital, Tongji University School of Medicine, Shanghai, China;Research Centre for Liver Failure, Beijing 302nd Hospital, Beijing, China;Research and Development Department, Beijing Center for Physical and Chemical Analysis, Beijing, China;State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China

关键词: Hepatitis B virus;    CRISPR-Cas9;    Integrated HBV DNA;    HBV cccDNA;    whole genome sequencing;   

DOI  :  10.3389/fcimb.2017.00091

学科分类：生物科学（综合）

来源: Frontiers

【 摘 要 】

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 analogues 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.

【 授权许可】

CC BY