CRISPR-Cas13a抑制RNA病毒的多靶标基因编辑技术构建  

Construction of Multi-target Editing Technology Based on CRISPR-Cas13a System to Inhibit RNA Viruses

作　　者：解屹 徐翔 葛明 张万红 黄坤[3] 江连强[4] 赵世民[5] 王玉洁 彭子忠 廖成 杨金广[1] 王凤龙[1] XIE Yi;XU Xiang;GE Ming;ZHANG Wanhong;HUANG Kun;JIANG Lianqiang;ZHAO Shimin;WANG Yujie;PENG Zizhong;LIAO Cheng;YANG Jinguang;WANG Fenglong(Institute of Tobacco Research of CAAS,Qingdao 266101,China;Graduate School of ChineseAcademy ofAgricultural Sciences,Beijing 100081,China;Honghe Prefecture Company of Yunnan Tobacco Company,Mile,Yunnan 652300;Liangshan Prefecture Company of Sichuan Tobacco Company,Xichang,Sichuan 615000,China;Luoyang Company of Henan Tobacco Company,Luoyang,Henan 471000,China;Enshi Tobacco Company Enshi Tobacco Branch,Enshi,Hubei 445000,China)

机构地区：[1]中国农业科学院烟草研究所,青岛266101 [2]中国农业科学院研究生院,北京100081 [3]云南省烟草公司红河州公司,云南弥勒652300 [4]四川省烟草公司凉山州公司,四川西昌615000 [5]河南省烟草公司洛阳市公司,河南洛阳471000 [6]恩施州烟草公司恩施市烟叶分公司,湖北恩施445000

出　　处：《中国烟草科学》2021年第6期36-44,共9页Chinese Tobacco Science

基　　金：中国烟草总公司烟草绿色防控重大专项[110202001033(LS-02)、110201901041(LS-04)];云南省烟草公司科技项目(2020530000241011);四川省烟草公司科技项目(SCYC202008);河南省烟草公司洛阳市公司科技项目(2020410300270076)。

摘　　要：CRISPR-Cas适应性免疫系统为细菌和古细菌免受外来核酸和噬菌体的侵害提供了保护。其中2类VI-A型CRISPR-Cas效应子Cas13a(之前称为C2c2),可受CRISPR RNA的引导,靶向切割与原始间隔区反向互补的单链RNA。本研究利用CRISPR-Lsh-Cas13a系统,多靶标编辑烟草花叶病毒(TMV)RdRP、MP和CP基因,以抑制病毒在寄主烟草体内的侵染与扩散,从而达到保护寄主的作用。该系统在瞬时表达测定中表现出对野生型TMV-U1和表达绿色荧光蛋白的TMV-30b的干扰以及对病毒累积和病毒病症状的抑制。靶向TMV RdRP基因的CRISPR RNA表现出比靶向MP和CP基因更好的编辑效能。经过适当设计的CRISPR-Lsh-Cas13a系统被赋予了广谱抗性,可以特异性编辑多种TMV菌株。研究表明,CRISPR-Cas13a系统可以用于针对RNA病毒的抑制干扰。The CRISPR-Cas adaptive immune system provides protection for bacteria and archaea from foreign nucleic acids and bacteriophages.In the CRISPR-Cas adaptive immune system,two classes of the VI-A CRISPR-Cas effector Cas13a(previously called C2c2)can be guided by CRISPR RNA to target and then cut the single-stranded RNA that is reversely complementary to the protospacers.In this study,the CRISPR-Lsh-Cas13a system was used for the multi-target editing of RdRP,MP,and CP genes of Tobacco mosaic virus(TMV)to protect the host from infection and spread of the virus.This system showed interference with wild-type TMV-U1 and TMV-30b expressing green fluorescent proteins in transient expression assays,as well as suppression of virus accumulation and viral disease symptoms.The CRISPR RNA targeting TMV RdRP gene showed better editing efficiency than that targeting MP and CP genes.The appropriately designed CRISPR-Lsh-Cas13a system was endowed with broad-spectrum resistance and can specifically edit a variety of TMV strains.This study showed that the CRISPR-Cas13a system can be used for the suppression and interference against RNA viruses.

关 键 词：基因编辑 CRISPR-Cas13a 烟草花叶病毒 病毒防控 多靶标编辑 

分 类 号：S432.41[农业科学—植物病理学]