外泌体可介导CRISPR/Cas9系统靶向切割乙型肝炎病毒基因组功能的细胞间传递  

Exosome-mediated CRISPR/Cas9 system targets to cut the intercellular transmission function of hepatitis B virus genome

作　　者：王杰[1] 陈然 鲁凤民[1] Wang Jie;Chen Ran;Lu Fengmin(Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China)

机构地区：[1]北京大学基础医学院病原生物学系暨感染病中心,100191

出　　处：《中华肝脏病杂志》2019年第8期610-614,共5页Chinese Journal of Hepatology

基　　金：国家"艾滋病和病毒性肝炎等重大传染病防治"科技重大专项"十三五"计划(2017ZX10202202、2017ZX10202203);北京市自然科学基金资助项目(7182080).

摘　　要：目的明确转染了规律间隔成簇短回文重复序列及其相关蛋白9(CRISPR/Cas9)表达质粒的细胞所分泌的外泌体中是否存在单链向导RNA(gRNA)和Cas9蛋白,并探索CRISPR/Cas9系统能否通过外泌体的细胞间传递,实现对周围细胞靶基因的编辑。方法(1)将CRISPR/Cas9表达质粒转染至HuH7细胞中,收集细胞培养上清液,差速离心法浓缩和纯化外泌体,通过电镜和马尔文激光散射粒度测定仪分别测定外泌体的形态和粒径,并利用逆转录-聚合酶链反应(RT-PCR)和蛋白质印迹法分别检测gRNA和Cas9蛋白水平。(2)将分别转染1.2×乙型肝炎病毒(HBV)表达质粒和HBV特异性CRISPR/Cas9表达质粒的HuH7细胞进行共培养。2d后提取细胞内HBVDNA,经PCR扩增后测序。结果转染CRISPR/Cas9表达质粒的HuH7细胞所产生的外泌体中不仅存在全长gRNA和Cas9蛋白,并可在细胞间传递其基因编辑功能,实现对周围细胞HBV基因组的破坏。结论作为一种潜在的递送系统,外泌体可通过携带有功能的gRNA和Cas9蛋白在细胞间传递CRISPR/Cas9系统的基因编辑功能。这一现象也提示我们,在利用CRISPR/Cas9系统进行基因治疗的过程中,也需要考虑携带有gRNA和Cas9蛋白的外泌体对周围及远端组织细胞的潜在影响。Objective To determine whether single-stranded guided RNA (gRNA) and Cas9 protein exist in the exosome secreted by cells transfected with CRISPR/Cas9 expression plasmid. Furthermore, to explore whether CRISPR/Cas9 system can edit target genes of peripheral cells through the intercellular transmission of exosomes. Methods (1) The CRISPR/Cas9 expression plasmid was transfected into HuH7 cells. The supernatant was collected and the exosomes were concentrated and purified by differential centrifugation. The morphology and particle size of exosomes were determined by electronic microscopy and Malvern laser scatter granulometry. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the levels of gRNA and Cas9 protein.(2) HuH7 cells transfected with pBB4.5 1.2×HBV and HBV specific CRISPR/Cas9 expression plasmids were co-cultured. After 2 days, HBV DNA was extracted and sequenced by PCR. Results There were not only full-length gRNA and Cas9 protein in the exosomes of Huh7 cells transfected with CRISPR / Cas9 expression plasmid. In addition, gene-editing functions were delivered between the cells to achieve the destruction of HBV genome of surrounding cells. Conclusion The CRISPR-Cas9 gene-editing system has the potential to deliver exosomes between cells via carrying functional gRNA and Cas9 proteins. This phenomenon hints that we are in the process of using the CRISPR/Cas9 system for gene therapy. Therefore, it is necessary to consider the potential effects of exosomes-carried gRNA and Cas9 proteins on the surrounding cells of the distal tubules.

关 键 词：肝炎病毒 乙型 CRISPR/Cas9 基因治疗 外泌体 递送系统 

分 类 号：R373.2[医药卫生—病原生物学]