通过CRISPR / Cas9介导的非切割碱基编辑永久灭活HBV基因组。

StephenW

Mol Ther Nucleic Acids. 2020 Mar 19;20:480-490. doi: 10.1016/j.omtn.2020.03.005. [Epub ahead of print]
Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing.
Yang YC1, Chen YH1, Kao JH2, Ching C1, Liu IJ3, Wang CC4, Tsai CH4, Wu FY1, Liu CJ2, Chen PJ2, Chen DS2, Yang HC5.
Author information

1
    Department of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan.
2
    Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Hepatitis Research Center, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Medical Research, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan.
3
    Department of Nursing, Cardinal Tien Junior College of Healthcare and Management, New Taipei City, Taiwan.
4
    Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan.
5
    Department of Microbiology, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei, Taiwan. Electronic address: [email protected].

Abstract

Current antiviral therapy fails to cure chronic hepatitis B virus (HBV) infection because of persistent covalently closed circular DNA (cccDNA). CRISPR/Cas9-mediated specific cleavage of cccDNA is a potentially curative strategy for chronic hepatitis B (CHB). However, the CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks (DSBs) of host genome, bearing the risk of genomic rearrangement and damage. Herein, we examined the utility of recently developed CRISPR/Cas-mediated "base editors" (BEs) in inactivating HBV gene expression without cleavage of DNA. Candidate target sites of the SpCas9-derived BE and its variants in HBV genomes were screened for generating nonsense mutations of viral genes with individual guide RNAs (gRNAs). SpCas9-BE with certain gRNAs effectively base-edited polymerase and surface genes and reduced HBV gene expression in cells harboring integrated HBV genomes, but induced very few insertions or deletions (indels). Interestingly, some point mutations introduced by base editing resulted in simultaneous suppression of both polymerase and surface genes. Finally, the episomal cccDNA was successfully edited by SpCas9-BE for suppression of viral gene expression in an in vitro HBV infection system. In conclusion, Cas9-mediated base editing is a potential strategy to cure CHB by permanent inactivation of integrated HBV DNA and cccDNA without DSBs of the host genome.

Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.

PMID:
    32278307
DOI:
    10.1016/j.omtn.2020.03.005

StephenW

摩尔的核酸。 2020 Mar 19; 20：480-490。 doi：10.1016 / j.omtn.2020.03.005。 [Epub提前发行]
通过CRISPR / Cas9介导的非切割碱基编辑永久灭活HBV基因组。
杨YC1，陈YH1，高JH2，程C1，刘IJ3，王CC4，蔡CH4，吴FY1，刘CJ2，陈PJ2，陈DS2，杨HC5。
作者信息

1个
    国立台湾大学医学院附属国立台湾大学医学院微生物系，台湾台北。
2
    国立台湾大学医学院国立台湾大学医学院临床医学研究所，台湾台北；台湾台北大学国立台湾大学医学院内科台湾台北国立台湾大学医学院肝炎研究中心，台湾台北；国立台湾大学医学院国立台湾大学医学院医学研究系，台湾台北。
3
    台湾新北市田心红初级卫生与管理学院护理系。
4
    国立台湾大学医学院临床医学研究所，国立台湾大学附属医院，台湾台北。
5
    国立台湾大学医学院附属国立台湾大学医学院微生物系，台湾台北；国立台湾大学医学院国立台湾大学医学院临床医学研究所，台湾台北；国立台湾大学医学院附属内科，国立台湾大学附属医院，台湾台北。电子地址：[email protected]。

抽象

由于持续的共价闭合环状DNA（cccDNA），当前的抗病毒治疗无法治愈慢性乙型肝炎病毒（HBV）感染。 CRISPR / Cas9介导的cccDNA特异性切割是治疗慢性乙型肝炎（CHB）的潜在方法。然而，CRISPR / Cas系统不可避免地靶向整合的HBV DNA，并诱导宿主基因组的双链断裂（DSB），承担着基因组重排和损坏的风险。本文中，我们研究了最近开发的CRISPR / Cas介导的“碱基编辑器”（BEs）在不切割DNA的情况下灭活HBV基因表达的效用。筛选了SpCas9衍生的BE及其在HBV基因组中的变体的候选靶位点，以产生带有单个指导RNA（gRNA）的病毒基因的无义突变。具有某些gRNA的SpCas9-BE可有效地编辑聚合酶和表面基因，并在包含整合的HBV基因组的细胞中降低HBV基因的表达，但几乎不会引起插入或缺失（indels）。有趣的是，碱基编辑引入的一些点突变导致同时抑制聚合酶和表面基因。最后，由SpCas9-BE成功编辑了游离cccDNA，以抑制体外HBV感染系统中病毒基因的表达。总之，Cas9介导的碱基编辑是通过永久灭活整合的HBV DNA和cccDNA而没有宿主基因组DSB的方法来治愈CHB的潜在策略。

版权所有©2020作者。由Elsevier Inc.出版。保留所有权利。

PMID：
    32278307
DOI：
    10.1016 / j.omtn.2020.03.005

StephenW

https://www.cell.com/molecular-t ... 62%3Fshowall%3Dtrue

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国立台湾大学

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跟俄罗斯的基因编辑，吉利德的原理差不多，都还没上临床