目的:探讨成簇的、规律间隔的短回文重复序列-相关核酸酶9(clustered regularly interspaced short palindromic repeats associated protein-9 nuclease,CRISPR-Cas9)技术的抗乙型肝炎病毒(hepatitis B virus,HBV)作用。方法:通过生物信息学筛选,获得12条靶向HBV基因组不同区域的sg RNA(single-guide RNA,sg RNA)并分别构建psp Cas9-sg RNA质粒,与HBV1.3倍体复制质粒共转染至Hep G2细胞,通过荧光定量PCR筛选能够有效抑制HBV复制的sg RNA,并使用Southern blot验证获得的sg RNA功能;使用最新报道的重组共价闭合环状DNA(recombinant covalently closed circular DNA,rccc DNA)模型评估CRISPRCas9对HBV共价闭合环状DNA(covalently closed circular DNA,ccc DNA)的抑制水平,并通过克隆测序检测rccc DNA的突变状况;对C57小鼠进行尾静脉高压水动力注射(hydrodynamic injection,HDI)rccc DNA质粒与psp Cas9-sg RNA,检测不同时间点外周血中乙型肝炎病毒s抗原(hepatitis B virus surface antigen,HBs Ag)及e抗原(hepatitis B virus e antigen,HBe Ag)水平,通过免疫组化技术观察CRISPR-Cas9处理后肝脏中乙型肝炎病毒核心抗原(hepatitis B virus core antigen,HBc Ag)水平,在动物水平评估CRISPR-Cas9对HBV复制的抑制作用。结果:所筛选的靶向乙型肝炎病毒基因组聚合酶编码区以及衣壳蛋白编码区的sg RNA5及sg RNA9能有效抑制细胞中HBV复制,分别使细胞复制模型中rc DNA降低至对照组的(43.64±4.66)%(P=0.000)和(44.86±2.25)%(P=0.000);在r CCCDNA细胞模型中证实2组sg RNA分别使ccc DNA降低至对照组的(29.39±3.18)%(P=0.000)及(26.83±1.76)%(P=0.000);CRISPR-Cas9系统主要使rccc DNA的靶点区域发生以短片段缺失为主的突变,突变率位50%~60%。CRISPR-Cas9系统能够有效降低HBV水动力小鼠模型血清HBs Ag(sg RNA5:P=0.000;sg RNA9:P=0.001)及HBe Ag浓度(sg RNA5:P=0.000;sg RNA9:P=0.000);小鼠血清HBV DNA水平与对照组相比降低约1个lg值(sg RNA5:P=0.000;sg RNA9:P=0.001),肝脏免疫组化结果显示CRISPR-Cas9系统能够降低组织HBc Ag表达。结论:CRISPR-Cas9系统作为一种基因编辑技术,能够在细胞水平及动物模型中发挥抗HBV作用,可以作为一种新型的抗病毒策略并为HBV感染的治疗提供了一个新的方向。 OBJECTIVE: To investigate the effects of clustered regularly spaced interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9) on hepatitis B virus (HBV) )effect. METHODS: Twelve sg RNAs targeting different regions of the HBV genome were obtained by bioinformatics screening and psp Cas9-sg RNA plasmids were constructed respectively and co-transfected with HBV1.3 ploidy plasmids To Hep G2 cells, sg RNAs that could effectively inhibit HBV replication were screened by fluorescence quantitative PCR and the sg RNA function was verified by Southern blot. Using the newly reported recombinant covalently closed circular DNA (rccc DNA ) Model was used to evaluate the inhibitory effect of CRISPRCas9 on the covalently closed circular DNA (ccc DNA). The mutation status of rccc DNA was detected by cloning and sequencing. The C57 mice were subjected to hydrodynamic injection , HDI) rccc DNA plasmid and psp Cas9-sg RNA were used to detect the levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus (HBeAg) in peripheral blood at different time points The levels of hepatitis B virus core antigen (HBcAg) in liver after CRISPR-Cas9 treatment were observed by immunohistochemistry. CRISPR-Cas9 Inhibition of HBV replication. Results: The selected sg RNA5 and sg RNA9 targeted to the coding region of HBV genomic polymerase and the coding region of capsid protein could effectively inhibit the HBV replication in the cell, and decreased the rc DNA in the cell replication model to that in the control group 43.64 ± 4.66)% and (44.86 ± 2.25)%, respectively (P = 0.000). In r CCCDNA cell model, the two groups of sg RNAs decreased ccc DNA to (29.39 ± 3.18)% P = 0.000) and (26.83 ± 1.76)%, respectively (P = 0.000). The CRISPR-Cas9 system mainly caused the deletion of the short fragment in the target region of rccc DNA. The mutation rate ranged from 50% to 60%. CRISPR-Cas9 system can effectively reduce the serum HBsAg (sg RNA5: P = 0.000; sg RNA9: P = 0.001) and HBeAg concentration (sg RNA5: ; Serum HBV DNA levels in mice decreased by about 1 lg compared with the control group (sg RNA5: P = 0.000; sg RNA9: P = 0.001). Immunohistochemical results showed that CRISPR-Cas9 system could reduce the expression of HBc Ag . Conclusion: As a gene editing technology, CRISPR-Cas9 system can play an anti-HBV role in cell level and animal models, and can be used as a new antiviral strategy and provide a new direction for the treatment of HBV infection.