Targeting hepatitis B virus with CRISPR-Cas9 efficiently inhibited virus replication

Deng Wanyan; Meng Tian; Qian Keli; Cai Xuefei; Deng Haijun; Hu Jieli; Huang Ailong; Long Quanxin

doi:10.13406/j.cnki.cyxb.001380

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Targeting hepatitis B virus with CRISPR-Cas9 efficiently inhibited virus replication

更新时间：2024-01-17

- Targeting hepatitis B virus with CRISPR-Cas9 efficiently inhibited virus replication
- Journal of Chongqing Medical University Vol. 42, Issue 11, Pages: 1514-1521(2017)
- 作者机构：
  
  1. 重庆医科大学附属第二医院感染性疾病分子生物学教育部重点实验室肝病研究所
  2. 重庆市第五人民医院感染科
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.001380
  CLC： R373.21
- Published：2017，
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Deng Wanyan, Meng Tian, Qian Keli, et al. Targeting hepatitis B virus with CRISPR-Cas9 efficiently inhibited virus replication. [J]. Journal of Chongqing Medical University 42(11):1514-1521(2017)
DOI：

Deng Wanyan, Meng Tian, Qian Keli, et al. Targeting hepatitis B virus with CRISPR-Cas9 efficiently inhibited virus replication. [J]. Journal of Chongqing Medical University 42(11):1514-1521(2017) DOI： 10.13406/j.cnki.cyxb.001380.

摘要

目的:探讨成簇的、规律间隔的短回文重复序列-相关核酸酶9（clustered regularly interspaced short palindromic repeats associated protein-9 nuclease

CRISPR-Cas9）技术的抗乙型肝炎病毒（hepatitis B virus

HBV）作用。方法:通过生物信息学筛选

获得12条靶向HBV基因组不同区域的sg RNA（single-guide RNA

sg RNA）并分别构建psp Cas9-sg RNA质粒

与HBV1.3倍体复制质粒共转染至Hep G2细胞

通过荧光定量PCR筛选能够有效抑制HBV复制的sg RNA

并使用Southern blot验证获得的sg RNA功能;使用最新报道的重组共价闭合环状DNA（recombinant covalently closed circular DNA

rccc DNA）模型评估CRISPRCas9对HBV共价闭合环状DNA（covalently closed circular DNA

ccc DNA）的抑制水平

并通过克隆测序检测rccc DNA的突变状况;对C57小鼠进行尾静脉高压水动力注射（hydrodynamic injection

HDI）rccc DNA质粒与psp Cas9-sg RNA

检测不同时间点外周血中乙型肝炎病毒s抗原（hepatitis B virus surface antigen

HBs Ag）及e抗原（hepatitis B virus e antigen

HBe Ag）水平

通过免疫组化技术观察CRISPR-Cas9处理后肝脏中乙型肝炎病毒核心抗原（hepatitis B virus core antigen

HBc Ag）水平

在动物水平评估CRISPR-Cas9对HBV复制的抑制作用。结果:所筛选的靶向乙型肝炎病毒基因组聚合酶编码区以及衣壳蛋白编码区的sg RNA5及sg RNA9能有效抑制细胞中HBV复制

分别使细胞复制模型中rc DNA降低至对照组的（43.64±4.66）%（P=0.000）和（44.86±2.25）%（P=0.000）;在r CCCDNA细胞模型中证实2组sg RNA分别使ccc DNA降低至对照组的（29.39±3.18）%（P=0.000）及（26.83±1.76）%（P=0.000）;CRISPR-Cas9系统主要使rccc DNA的靶点区域发生以短片段缺失为主的突变

突变率位50%

0%。CRISPR-Cas9系统能够有效降低HBV水动力小鼠模型血清HBs Ag（sg RNA5:P=0.000;sg RNA9:P=0.001）及HBe Ag浓度（sg RNA5:P=0.000;sg RNA9:P=0.000）;小鼠血清HBV DNA水平与对照组相比降低约1个lg值（sg RNA5:P=0.000;sg RNA9:P=0.001）

肝脏免疫组化结果显示CRISPR-Cas9系统能够降低组织HBc Ag表达。结论:CRISPR-Cas9系统作为一种基因编辑技术

能够在细胞水平及动物模型中发挥抗HBV作用

可以作为一种新型的抗病毒策略并为HBV感染的治疗提供了一个新的方向。

Abstract

Objective:To investigate the antivirus activity of HBV targeted CRISPR-Cas9 system. Methods:Twelve sg RNAs（singleguide RNA）that target to different hepatitis B virus（HBV）genome were designed by bioinformatic screening. After transient transfection experiment in Hep G2 cell

Southern blot and quantitative real time PCR were performed to prove the anti-HBV efficiency of each psp Cas9-sg RNA plasmid. A newly published rccc DNA cell model was employed to evaluate the activity of CRISPR-Cas9 in eliminating ccc DNA. The anti-HBV effects of CRISPRCas9 in vivo were evaluated in hydrodynamic injection mice model. Results:sg RNA 5 and sg RNA 9 targeted to polymerase and core genes of HBV genome were proved to be effective by transient transfection experiment in Hep G2 cell

rc DNA were reduced significantly（P=0.000

respectively）. CRISPR-Cas9 system with sg RNA5 and sg RNA9 significantly reduced ccc DNA level in rccc DNA cell model（P=0.000

respectively）

and the short fragment deletions nearby target sites were the major mutation phenotype

50% to 60% ccc DNA possessed these deletion characteristics. Sg RNA5 and sg RNA9 containing psp Cas9 plasmid can reduce HBs Ag（P=0.000

P=0.001

respectively）

HBe Ag（P=0.000

P=0.000

respectively）and HBV DNA level（P=0.000

P=0.001

respectively）in mice serum on day 3 and day 6 after injection effectively. Immunohistochemical detection of HBc Ag in the liver tissue of mice treated with sg RNA containing CRISPR-Cas9 plasmid revealed a decrease in measurable HBc Ag in hepatocytes. Conclusion:Targeting Hepatitis B virus with CRISPR-Cas9 is an effective therapeutic approach to control the virus replication.

关键词

乙型肝炎病毒成簇的、规律间隔的短回文重复序列-相关核酸酶9技术抗病毒作用

Keywords

hepatitis B virusclustered regularly interspaced short palindromic repeats associated protein-9 nucleaseantiviral

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