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Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle
Published on: February 1, 2017
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Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication
Jie Wang1, Zhong-Wei Xu1, Shuang Liu1
1Jie Wang, Rui-Yang Zhang, Shan-Long Ding, Lu Long, Xiang-Mei Chen, Hui Zhuang, Feng-Min Lu, State Key Laboratory of Natural and Biomimetic Drugs, Department of Microbiology and Infectious Disease Center, School of Basic Medicine, Peking University Health Science Center, Beijing 100191, China.
World Journal of Gastroenterology
|September 2, 2015
Summary
This study identified effective CRISPR/Cas9 guide RNAs (gRNAs) that suppress hepatitis B virus (HBV) replication and destroy cccDNA reservoirs. These dual-gRNAs show potential for treating chronic HBV infection with minimal cytotoxicity.
Area of Science:
- Molecular Biology
- Virology
- Gene Editing
Background:
- Hepatitis B virus (HBV) infection is a global health concern.
- Persistent HBV infection is characterized by the presence of covalently closed circular DNA (cccDNA).
- Current treatments do not fully eradicate HBV cccDNA.
Purpose of the Study:
- To screen and identify effective guide RNAs (gRNAs) targeting HBV genotypes A-D.
- To evaluate the efficacy of dual-gRNA combinations in suppressing HBV replication.
- To assess the potential of CRISPR/Cas9 for HBV cccDNA eradication.
Main Methods:
- Designed 15 gRNAs and 11 dual-gRNA combinations targeting HBV regulatory regions.
- Assessed HBsAg and HBeAg suppression in cell culture supernatants.
- Utilized PCR, sequencing, and KCl precipitation with PSAD digestion to confirm HBV template and cccDNA destruction.
- Evaluated gRNA cytotoxicity using a mitochondrial tetrazolium assay.
Main Results:
- All tested gRNAs significantly reduced HBsAg/HBeAg production.
- Dual-gRNAs demonstrated enhanced suppression of HBsAg/HBeAg compared to single gRNAs across HBV genotypes A-D.
- PCR sequencing confirmed specific destruction of HBV templates by dual-gRNAs.
- A specific dual-gRNA combination (gRNA-5 and gRNA-12) effectively reduced viral antigens and destroyed cccDNA reservoirs.
Conclusions:
- CRISPR/Cas9 system efficiently targets and destroys HBV expressing templates (genotypes A-D) without significant cytotoxicity.
- Dual-gRNAs, particularly the gRNA-5 and gRNA-12 combination, show promise in eliminating HBV cccDNA.
- This approach represents a potential strategy for eradicating persistent HBV cccDNA in chronic HBV infection.
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