The CRISPR/Cas9 system is a novel genome editing technology which has been successfully used to inhibit HBV replication. Here, we described a novel gRNA-microRNA (miRNA)-gRNA ternary cassette driven by a single U6 promoter. With an anti-HBV pri-miR31 mimic integrated between two HBV-specific gRNAs, both gRNAs could be separated from the long transcript of gRNA-miR-HBV-gRNA ternary cassette through Drosha/DGCR8 processing. The results showed that the gRNA-miR-HBV-gRNA ternary cassette could efficiently express two gRNAs and miR-HBV. The optimal length of pri-miRNA flanking sequence in our ternary cassette was determined to be 38 base pairs (bp). Besides, HBV-specific gRNAs and miR-HBV in gRNA-miR-HBV-gRNA ternary cassette could exert a synergistic effect in inhibiting HBV replication and destroying HBV genome in vitro and in vivo. Most importantly, together with RNA interference (RNAi) approach, the HBV-specific gRNAs showed the potent activity on the destruction of HBV covalently closed circular DNA (cccDNA). Since HBV cccDNA is an obstacle for the elimination of chronic HBV infection, the gRNA-miR-HBV-gRNA ternary cassette may be a potential tool for the clearance of HBV cccDNA.

中文翻译：

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结合 CRISPR/Cas9 和 RNAi 方法的 gRNA-miRNA-gRNA 三元盒强烈抑制乙型肝炎病毒复制


CRISPR/Cas9系统是一种新型基因组编辑技术，已成功用于抑制乙型肝炎病毒复制。在这里，我们描述了一种由单个 U6 启动子驱动的新型 gRNA-microRNA (miRNA)-gRNA 三元盒。通过在两个 HBV 特异性 gRNA 之间集成抗 HBV pri-miR31 模拟物，可以通过 Drosha/DGCR8 处理将这两个 gRNA 从 gRNA-miR-HBV-gRNA 三元盒的长转录本中分离出来。结果表明，gRNA-miR-HBV-gRNA三元盒可以有效表达两种gRNA和miR-HBV。我们的三元盒中 pri-miRNA 侧翼序列的最佳长度被确定为 38 个碱基对 (bp)。此外，gRNA-miR-HBV-gRNA三元盒中的HBV特异性gRNA和miR-HBV可以在体内外抑制HBV复制和破坏HBV基因组方面发挥协同作用。最重要的是，结合 RNA 干扰 (RNAi) 方法，HBV 特异性 gRNA 对破坏 HBV 共价闭合环状 DNA (cccDNA) 表现出强大的活性。由于HBV cccDNA是消除慢性HBV感染的障碍，因此gRNA-miR-HBV-gRNA三元盒可能是清除HBV cccDNA的潜在工具。