title: An Effective Molecular Target Site in Hepatitis B Virus S Gene for Cas9 Cleavage and Mutational Inactivation.
abstract: Chronic hepatitis B infection remains incurable because HBV cccDNA can persist indefinitely in patients recovering from acute HBV infection. Given the incidence of HBV infection and the shortcomings of current therapeutic options, a novel antiviral strategy is urgently needed. To inactivate HBV replication and destroy the HBV genome, we employed genome editing tool CRISPR/Cas9. Specifically, we found a CRISPR/Cas9 system (gRNA-S4) that effectively targeted the HBsAg region and could suppress efficiently viral replication with minimal off-target effects and impact on cell viability. The mutation mediated by CRISPR/Cas9 in HBV DNA both in a stable HBV-producing cell line and in HBV transgenic mice had been confirmed and evaluated using deep sequencing. In addition, we demonstrated the reduction of HBV replication was caused by the mutation of S4 site through three S4 region-mutated monoclonal cells. Besides, the gRNA-S4 system could also reduce serum surface-antigen levels by 99.91 ± 0.05% and lowered serum HBV DNA level below the negative threshold in the HBV hydrodynamics mouse model. Together, these findings indicate that the S4 region may be an ideal target for the development of innovative therapies against HBV infection using CRISPR/Cas9.
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Hao
authors: H
authors: Li
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Chunyu
authors: C
authors: Sheng
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Hongbo
authors: H
authors: Liu
authors: Transgenic Engineering Research Laboratory, Infectious Disease Center, Guangzhou 458 th Hospital, Guangzhou, China.
authors: Guangze
authors: G
authors: Liu
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Xinying
authors: X
authors: Du
authors: Lab of Blood-Borne Viruses, Beijing Institute of Transfusion Medicine, Beijing, China.
authors: Juan
authors: J
authors: Du
authors: Lab of Blood-Borne Viruses, Beijing Institute of Transfusion Medicine, Beijing, China.
authors: Linsheng
authors: L
authors: Zhan
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Peng
authors: P
authors: Li
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Chaojie
authors: C
authors: Yang
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Lihua
authors: L
authors: Qi
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Jian
authors: J
authors: Wang
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Xiaoxia
authors: X
authors: Yang
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Leili
authors: L
authors: Jia
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Jing
authors: J
authors: Xie
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Ligui
authors: L
authors: Wang
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Rongzhang
authors: R
authors: Hao
authors: Research Center for Liver Failure, Beijing 302 nd Hospital, Beijing, China.
authors: Dongping
authors: D
authors: Xu
authors: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
authors: Yigang
authors: Y
authors: Tong
authors: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
authors: Yusen
authors: Y
authors: Zhou
authors: Gladcan Consulting Company, Beijing, China.
authors: Jianjun
authors: J
authors: Zhou
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Yansong
authors: Y
authors: Sun
authors: Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA.
authors: Qiao
authors: Q
authors: Li
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Shaofu
authors: S
authors: Qiu
authors: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors: Hongbin
authors: H
authors: Song
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D064113:CRISPR-Cas Systems / Q000187:drug effects / Q000235:genetics*; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics* / Q000502:physiology*; D009154:Mutation / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics / Q000502:physiology*
chemical_list: D000998:Antiviral Agents; D014764:Viral Proteins
keywords: CRISPR/Cas9; HBV infection