摘要
目的应用近年发展的基因编辑技术成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeat，CRISPR）引导的Cas9核酸酶特异性靶向切割HBVDNA基因。方法基于慢病毒载体的lentiCRISPRv2系统，我们设计了3个特异靶向HBV基因组的向导RNA序列（guideRNA，gRNA）。质粒pUC18-HBV1．2和lentiCRISPR-gRNAs共转染HepG2细胞，或lentiCRISPR-gRNAs与相关慢病毒载体转染293T细胞后产生相应慢病毒。用慢病毒处理整合了HBVDNA的HepG2．2．15细胞或者HepDE19细胞系：并用慢病毒处理基于C57BL／6小鼠腺相关病毒HBV复制模型。结果单靶向CRISPR／Cas9切割可以抑制30％～50％病毒蛋白产生。两个或三个靶向切割则显著提高抑制效率，可达70％-90％。以慢病毒体系处理HepG2．2．15或HepDES19细胞时，HepDES19细胞中HBeAg下降幅度约为HepG2．2．15细胞中的3倍。HBV复制小鼠模型在CRISPR／Cas9慢病毒处理后，血清中HBsAg和HBeAg可转为阴性。结论CRISPR／Cas9系统多位点靶向切割可高效清除细胞内基因组，相对于整合于染色体的基因组，游离于细胞核的HBV基因组对此靶向切割系统更加敏感。
Objective To specifically cleave the intraceUular HBV DNA using the clustered regularly interspaced short palindromic repeat （CRISPR）/Cas9 genome editing technology. Methods Using of Lenti CRISPRv2 system, we designed 3 highly conserved guide RNAs （gRNAs） to specifically cleave HBV genome. The cleavage efficacy of CRISPR/Cas9 targeting at HBV DNA in plasmids, the episomal HBV DNA or integrated HBV DNA in chromosome was evaluated in the cell cultures of HepG2. 2. 15 and HepDES19, and in a model of C57BL/6 mouse infected with AAV-HBV1.3. Results Viral proteins were inhibited by 30% - 50% by a single site CRISPR/Cas9 cleavagein the HepG2 cells transiently co-transfected with HBV DNA, multiple sites of cleavage significantly elevated the efficiency to 70% - 90%. Compared with that of HepG2. 2. 15 cells, HBeAg in I-IepDES19 cells was inhibitedby at least three-fold higher. Conclusion Multiple sites cleavage significantly improve the efficacy of CRISPR/Cas9 systems, and this system is more efficient to cleave the extra-chromosome HBV DNA than the integrated HBV DNA in chromosomes.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2016年第1期53-57,共5页
Chinese Journal of Experimental and Clinical Virology
基金
国家自然科学基金（81370540）
