Yang Yu-Chan, Chen Yu-Hsiang, Kao Jia-Horng, Ching Chi, Liu I-Jung, Wang Chih-Chiang, Tsai Cheng-Hsueh, Wu Fang-Yi, Liu Chun-Jen, Chen Pei-Jer, Chen Ding-Shinn, Yang Hung-Chih
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; 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.
Mol Ther Nucleic Acids. 2020 Jun 5;20:480-490. doi: 10.1016/j.omtn.2020.03.005. Epub 2020 Mar 19.
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.
由于共价闭合环状DNA（cccDNA）持续存在，目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特异性切割是慢性乙型肝炎（CHB）潜在的治愈策略。然而，CRISPR/Cas系统不可避免地靶向整合的HBV DNA并诱导宿主基因组双链断裂（DSB），存在基因组重排和损伤的风险。在此，我们研究了最近开发的CRISPR/Cas介导的“碱基编辑器”（BE）在不切割DNA的情况下使HBV基因表达失活的效用。筛选了SpCas9衍生的BE及其变体在HBV基因组中的候选靶位点，以通过单个向导RNA（gRNA）产生病毒基因的无义突变。带有特定gRNA的SpCas9-BE有效地对聚合酶和表面基因进行碱基编辑，并降低了含有整合HBV基因组的细胞中的HBV基因表达，但诱导的插入或缺失（indel）很少。有趣的是，碱基编辑引入的一些点突变导致聚合酶和表面基因同时受到抑制。最后，在体外HBV感染系统中，SpCas9-BE成功编辑了游离cccDNA以抑制病毒基因表达。总之，Cas9介导的碱基编辑是一种通过永久使整合的HBV DNA和cccDNA失活而不产生宿主基因组DSB来治愈CHB的潜在策略。
