Efficient inhibition of duck hepatitis B virus DNA by the CRISPR/Cas9 system

Zheng,Qingfen; Bai,Li; Zheng,Sujun; Liu,Mei; Zhang,Jinyan; Wang,Ting; Xu,Zhongwei; Chen,Yu; Li,Jiansheng; Duan,Zhongping

doi:10.3892/mmr.2017.7518

Efficient inhibition of duck hepatitis B virus DNA by the CRISPR/Cas9 system

Authors:
- Qingfen Zheng
- Li Bai
- Sujun Zheng
- Mei Liu
- Jinyan Zhang
- Ting Wang
- Zhongwei Xu
- Yu Chen
- Jiansheng Li
- Zhongping Duan
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Affiliations: Artificial Liver Center, Beijing Youan Hospital, Capital Medical University, Beijing 100069, P.R. China, Department of Gastroenterology, Pennsylvania Hospital, University of Pennsylvania, Philadelphia, PA 19104, USA, Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China
Published online on: September 19, 2017 https://doi.org/10.3892/mmr.2017.7518
Pages: 7199-7204
Copyright: © Zheng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Current therapeutic strategies cannot eradicate hepatitis B virus covalently closed circular DNA (HBV cccDNA), which accounts for the persistence of HBV infection. Very recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‑associated protein 9 (Cas9) system has been used as an efficient and powerful tool for viral genome editing. Given that the primary duck hepatocyte (PDH) infected with duck hepatitis B virus (DHBV) has been widely used to study human HBV infection in vitro, the present study aimed to demonstrate the targeted inhibition of DHBV DNA, especially cccDNA, by the CRISPR/Cas9 system using this model. We designed six single‑guide RNAs (sgRNA1‑6) targeting the DHBV genome. The sgRNA/Cas9 plasmid was transfected into DHBV‑infected PDHs, and then DHBV total DNA (in culture medium and PDHs) and cccDNA were quantified by reverse transcription‑quantitative polymerase chain reaction. The combined inhibition of CRISPR/Cas9 system and entecavir (ETV) was also assessed. Two sgRNAs, sgRNA4 and sgRNA6, exhibited efficient inhibition on DHBV total DNA (77.23 and 86.51%, respectively), cccDNA (75.67 and 85.34%, respectively) in PDHs, as well as DHBV total DNA in the culture medium (62.17 and 59.52%, respectively). The inhibition remained or enhanced from day 5 to day 9 following transfection. The combination of the CRISPR/Cas9 system and ETV further increased the inhibitory effect on DHBV total DNA in PDHs and culture medium, but not cccDNA. The CRISPR/Cas9 system has the potential to be a useful tool for the suppression of DHBV DNA.

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