On April 3, 2015, researchers at Sidong Xiong’s group published an article entitled “Targeting hepatitis B virus cccDNA by CRISPR/Cas9 nuclease efficiently inhibits viral replication” in Antiviral Research.
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-a 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.
Prof. Chunsheng Dong and Liang Qu are the first authors of the paper. Contributing authors are Lin Wei and Yuanshu Dong. Prof. Sidong Xiong and Prof. Chunsheng Dong are the corresponding authors of the paper. This work has been supported by the grants from Major State Basic Research Development Program of China (2013CB531502, 2013CB530501); the National Natural Science Foundation of China (31270977, 81072413, 81101257, 31170878); The Scientific Research Foundation for Returned Overseas Chinese Scholars from State Education Ministry to C.D.; Jiangsu “Pan-Deng” Project (BK2010004); Jiangsu “333” project of cultivation of high-level talents; Qing Lan Project of the Jiangsu higher education institutions;Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD); Jiangsu Provincial Innovative Research Team and Jiangsu Students’ Platform for innovation and entrepreneurship training program 201310285046Z.