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通过 CRISPR 介导的碱基编辑有效沉默乙型肝炎病毒 S 基因

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发表于 2022-3-29 19:27 |显示全部帖子
通过 CRISPR 介导的碱基编辑有效沉默乙型肝炎病毒 S 基因
周浩 1 2 , 王晓梅 1 2 , Clifford J Steer 2 , 宋桂生 2 , 牛俊奇 1
隶属关系
隶属关系

    1
    【作者单位】: 吉林大学第一医院肝内科;
    2
    美国明尼苏达州明尼阿波利斯市明尼苏达大学医学院医学系。

    PMID:35338607 DOI:10.1002/hep4.1933

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抽象的

乙型肝炎病毒(HBV)感染是肝硬化和肝细胞癌的主要危险因素。成簇的规则间隔短回文重复序列 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 已被用于精确编辑 HBV 基因组并通过双链断裂 (DSB) 的非同源末端连接修复来消除 HBV。然而,CRISPR/Cas9介导的DSB会引发宿主基因组的不稳定性,并且编辑基因组的效率较低,限制了其应用。 CRISPR 胞苷碱基编辑器 (CBE) 可以通过产生过早的终止密码子来沉默基因。在这里,我们开发了一种 CRISPR 碱基编辑器方法来精确编辑 HBV 基因组中的单个核苷酸以削弱 HBV 基因表达。具体而言,设计了一种单向导 RNA (sgRNA),用于编辑 HBV S 基因的第 30 个密码子,该基因编码 HBV 表面抗原 (HBsAg),从 CAG(谷氨酰胺)到终止密码子 TAG。我们接下来使用携带HBV基因组的人肝癌PLC/PRF/5细胞建立表达CBE的细胞系(PLC/PRF/5-CBE)。慢病毒用于将 sgRNA 引入 PLC/PRF/5-CBE 细胞。表型上,71% 的 PLC/PRF/5-CBE 细胞在 S 基因内产生了过早的终止密码子。 HBs 信使 RNA 水平显着降低。在 PLC/PRF/5-CBE 细胞中观察到 HBsAg 分泌减少 92%。 gRNA_S 处理后,细胞内 HBsAg 也降低了 84%。此外,在 HBV 基因组内预测的脱靶基因座中未检测到脱靶效应。测序证实HBV基因型B、C、F、G和H的95%、93%、93%、9%和72%的S基因序列具有sgRNA的结合位点。结论:我们的研究结果表明,CRISPR 介导的碱基编辑是一种沉默 HBV S 基因的有效方法,表明其具有消除 HBV 的治疗潜力。

© 2022 作者。由 Wiley Periodicals LLC 代表美国肝病研究协会出版的 Hepatology Communications。

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发表于 2022-3-29 19:27 |显示全部帖子
Efficient silencing of hepatitis B virus S gene through CRISPR-mediated base editing
Hao Zhou  1   2 , Xiaomei Wang  1   2 , Clifford J Steer  2 , Guisheng Song  2 , Junqi Niu  1
Affiliations
Affiliations

    1
    Department of Hepatology, The First Hospital of Jilin University, Changchun, China.
    2
    Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA.

    PMID: 35338607 DOI: 10.1002/hep4.1933

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Abstract

Hepatitis B virus (HBV) infection is a major risk factor of liver cirrhosis and hepatocellular carcinoma. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been used to precisely edit the HBV genome and eliminate HBV through non-homologous end-joining repair of double-stranded break (DSB). However, the CRISPR/Cas9-mediated DSB triggers instability of host genome and exhibits low efficiency to edit genome, limiting its application. CRISPR cytidine base editors (CBEs) could silence genes by generating a premature stop codon. Here we developed a CRISPR base editor approach to precisely edit single nucleotide within the HBV genome to impair HBV gene expression. Specifically, a single-guide RNA (sgRNA) was designed to edit the 30th codon of HBV S gene, which encodes HBV surface antigen (HBsAg), from CAG (glutamine) to stop codon TAG. We next used human hepatoma PLC/PRF/5 cells carrying the HBV genome to establish a cell line that expresses a CBE (PLC/PRF/5-CBE). Lentivirus was used to introduce sgRNA into PLC/PRF/5-CBE cells. Phenotypically, 71% of PLC/PRF/5-CBE cells developed a premature stop codon within the S gene. Levels of HBs messenger RNA were significantly decreased. A 92% reduction of HBsAg secretion was observed in PLC/PRF/5-CBE cells. The intracellular HBsAg was also reduced by 84% after treatment of gRNA_S. Furthermore, no off-target effect was detected in predicted off-target loci within the HBV genome. Sequencing confirmed that 95%, 93%, 93%, 9%, and 72% S gene sequences of HBV genotypes B, C, F, G, and H had the binding site of sgRNA. Conclusion: Our findings indicate that CRISPR-mediated base editing is an efficient approach to silence the HBV S gene, suggesting its therapeutic potential to eliminate HBV.

© 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.

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发表于 2022-3-29 19:28 |显示全部帖子
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