The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo.
The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo.
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DOI：
10.1038/mtna.2014.38
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发表时间：
2014-08-19
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在当前抗病毒治疗下，乙型肝炎病毒（HBV）共价闭合环状DNA（cccDNA）的持续存在是根除慢性乙型肝炎（CHB）的主要障碍。治愈CHB将需要针对cccDNA特异性破坏的新策略。成簇规律间隔短回文重复序列（CRISPR）/Cas9系统是一种新开发的工具，可通过与目标DNA序列碱基配对的合成引导RNA（gRNA）对DNA目标进行位点特异性切割。为了检验该系统是否能够切割HBV基因组，我们针对A型HBV设计了8种gRNA。利用HBV特异性gRNA，CRISPR/Cas9系统显著降低了转染HBV表达载体的Huh - 7细胞中HBV核心蛋白和表面蛋白的产生。在筛选的8种gRNA中，确定了2种有效的gRNA。有趣的是，一种靶向保守HBV序列的gRNA可作用于不同基因型。利用 hydrodynamic - HBV持续感染小鼠模型，我们进一步证明该系统能够切割含有肝内HBV基因组的质粒，并促进其在体内的清除，从而导致血清表面抗原水平降低。这些数据表明，CRISPR/Cas9系统能够在体外和体内破坏HBV表达模板，显示其在根除持续性HBV感染方面的潜力。
Persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) under current antiviral therapy is a major barrier to eradication of chronic hepatitis B (CHB). Curing CHB will require novel strategies for specific disruption of cccDNA. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a newly developed tool for site-specific cleavage of DNA targets directed by a synthetic guide RNA (gRNA) base-paired to the target DNA sequence. To examine whether this system can cleave HBV genomes, we designed eight gRNAs against HBV of genotype A. With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector. Among eight screened gRNAs, two effective ones were identified. Interestingly, one gRNA targeting the conserved HBV sequence acted against different genotypes. Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.
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