Theranostics 2017; 7(12):3090-3105. doi:10.7150/thno.18114

Research Paper

The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication

Jie Wang1*, Ran Chen1*, Ruiyang Zhang1*, Shanlong Ding1, Tianying Zhang2, Quan Yuan2, Guiwen Guan1, Xiangmei Chen1, Ting Zhang1, Hui Zhuang1, Frederick Nunes3, Timothy Block4, Shuang Liu5, Zhongping Duan5, Ningshao Xia2✉, Zhongwei Xu3✉, Fengmin Lu1✉

1. State Key Laboratory of Natural and Biomimetic Drugs, Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China;
2. State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, China;
3. Department of Gastroenterology, Pennsylvania Hospital, University of Pennsylvania, Philadelphia, Pennsylvania, USA;
4. Baruch S. Blumberg Institute, Doylestown, Pennsylvania, USA;
5. Beijing Artificial Liver Treatment & Training Center, Beijing Youan Hospital, Capital Medical University, Beijing, China.
* These authors contributed equally to this work.


The CRISPR/Cas9 system is a novel genome editing technology which has been successfully used to inhibit HBV replication. Here, we described a novel gRNA-microRNA (miRNA)-gRNA ternary cassette driven by a single U6 promoter. With an anti-HBV pri-miR31 mimic integrated between two HBV-specific gRNAs, both gRNAs could be separated from the long transcript of gRNA-miR-HBV-gRNA ternary cassette through Drosha/DGCR8 processing. The results showed that the gRNA-miR-HBV-gRNA ternary cassette could efficiently express two gRNAs and miR-HBV. The optimal length of pri-miRNA flanking sequence in our ternary cassette was determined to be 38 base pairs (bp). Besides, HBV-specific gRNAs and miR-HBV in gRNA-miR-HBV-gRNA ternary cassette could exert a synergistic effect in inhibiting HBV replication and destroying HBV genome in vitro and in vivo. Most importantly, together with RNA interference (RNAi) approach, the HBV-specific gRNAs showed the potent activity on the destruction of HBV covalently closed circular DNA (cccDNA). Since HBV cccDNA is an obstacle for the elimination of chronic HBV infection, the gRNA-miR-HBV-gRNA ternary cassette may be a potential tool for the clearance of HBV cccDNA.

Keywords: CRISPR/Cas9, miRNA, gRNA-miRNA-gRNA ternary cassette, HBV, cccDNA.

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How to cite this article:
Wang J, Chen R, Zhang R, Ding S, Zhang T, Yuan Q, Guan G, Chen X, Zhang T, Zhuang H, Nunes F, Block T, Liu S, Duan Z, Xia N, Xu Z, Lu F. The gRNA-miRNA-gRNA Ternary Cassette Combining CRISPR/Cas9 with RNAi Approach Strongly Inhibits Hepatitis B Virus Replication. Theranostics 2017; 7(12):3090-3105. doi:10.7150/thno.18114. Available from