title: Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication.
abstract: Chronic hepatitis B virus (HBV) infection causes liver cirrhosis and hepatocellular carcinoma and remains a serious health problem worldwide. Covalently closed circular DNA (cccDNA) in the liver cell nucleus sustains HBV infection. Major treatments for HBV infection include the use of interferon-α and nucleotide analogs, but they cannot eradicate cccDNA. As a novel tool for genome editing, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system developed from bacteria can be used to accurately and efficiently engineer and modify genomic DNA. In this study, the CRISPR/Cas9 system was used to target the HBV genome and efficiently inhibit HBV infection. We synthesized four single-guide RNAs (sgRNAs) targeting the conserved regions of HBV. The expression of these sgRNAS with Cas9 reduced the viral production in Huh7 cells as well as in HBV-replication cell HepG2.2.15. We further demonstrated that CRISPR/Cas9 direct cleavage and cleavage-mediated mutagenesis occurred in HBV cccDNA of transfected cells. In the new mouse model carrying HBV cccDNA, injection of sgRNA-Cas9 plasmids via rapid tail vein resulted in the low level of cccDNA and HBV protein. In conclusion, the designed CRISPR/Cas9 system can accurately and efficiently target HBV cccDNA and inhibit HBV replication. This system may be used as a novel therapeutic strategy against chronic HBV infection.
authors.affiliation: Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Science, Soochow University, Suzhou 215123, China. Electronic address: chunshengdong@suda.edu.cn.
authors.forename: Chunsheng
authors.initials: C
authors.lastname: Dong
authors.affiliation: Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Science, Soochow University, Suzhou 215123, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Qu
authors.affiliation: State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Haoyi
authors.initials: H
authors.lastname: Wang
authors.affiliation: Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Science, Soochow University, Suzhou 215123, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wei
authors.affiliation: Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Science, Soochow University, Suzhou 215123, China.
authors.forename: Yuansu
authors.initials: Y
authors.lastname: Dong
authors.affiliation: Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Science, Soochow University, Suzhou 215123, China. Electronic address: sdxiongfd@126.com.
authors.forename: Sidong
authors.initials: S
authors.lastname: Xiong
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000008:administration & dosage* / Q000378:metabolism; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004270:DNA, Circular / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004195:Disease Models, Animal; D004337:Drug Carriers / Q000008:administration & dosage; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006515:Hepatitis B virus / Q000187:drug effects* / Q000502:physiology; D019694:Hepatitis B, Chronic / Q000188:drug therapy*; D022781:Hepatocytes / Q000187:drug effects* / Q000821:virology; D006801:Humans; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000008:administration & dosage; D016896:Treatment Outcome; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D064130:CRISPR-Associated Proteins; D004270:DNA, Circular; D004279:DNA, Viral; D004337:Drug Carriers; D004720:Endonucleases
keywords: CRISPR/Cas9; DNA targeting; HBV; Viral replication