利用CRISPR/Cas13d抑制肠道病毒71型感染  

Inhibition of Enterovirus 71 Infection Using CRISPR/Cas13d System

作　　者：张晓晓 古天乐 吴朔 徐溪 王高文 遆新宇 张艳 ZHANG Xiaoxiao;GU Tianle;WU Shuo;XU Xi;WANG Gaowen;TI Xinyu;ZHANG Yan(Department of Respiratory and Critical Care Medicine,Xijing Hospital,Xi’an 710000,Shaanxi,China;Department of Microbiology,School of Basic Medicine,Air Force Medical University,Xi’an 710032,Shaanxi,China)

机构地区：[1]西京医院呼吸与危重症医学科,中国陕西西安710000 [2]空军军医大学基础医学院微生物与病原生物学教研室,中国陕西西安710032

出　　处：《生命科学研究》2025年第1期56-61,共6页Life Science Research

基　　金：国家自然科学基金资助项目(82302525);部队项目(21FYFH02)。

摘　　要：肠道病毒71型(enterovirus 71,EV71)感染会导致手足口病和危及生命的神经系统疾病,目前尚无针对EV71的抗病毒药物可用。为了利用成簇规则间隔的短回文重复序列及其相关蛋白(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein,CRISPR/Cas)系统抑制EV71感染,本研究选择CRISPR/Cas13d系统,构建U6启动子驱动的向导RNA(guide RNA,g RNA)、EF-1α启动子驱动的Cas13d和绿色荧光蛋白(green fluorescent protein,GFP)的表达载体;将不同g RNA质粒分别转染人胚肾细胞293T(HEK293T)后感染EV71,通过实时荧光定量PCR检测EV71 VP1 m RNA的含量,采用Western-blot检测EV71 VP1蛋白的表达量,利用流式细胞术检测GFP蛋白的含量,运用CCK-8法检测细胞活力。结果显示,在5个靶向EV71基因组不同位置的g RNA质粒中,有4个可以显著抑制EV71 VP1 m RNA和蛋白质表达,其中1个质粒具有较强的旁切活性,但是所有的质粒均没有表现出显著的细胞毒性。实验结果初步表明,可以利用CRISPR/Cas13d系统抑制EV71感染,但是需要选择合适的g RNA,以保障抗病毒作用和避免细胞毒性。Enterovirus 71(EV71)infection can cause hand,foot,and mouth disease and life-threatening neurological disorders.Currently,there are no antiviral drugs available against the virus.In order to utilize the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein(CRISPR/Cas)system to suppress EV71 infection,the CRISPR/Cas13d system was chosen,and plasmids containing a guide RNA(gRNA)driven by the U6 promoter and Cas13d and green fluorescent protein(GFP)driven by the EF-1琢promoter were constructed.Human embryonic kidney cells(HEK293T)were transfected with a resultant plasmid,and then infected with EV71.The EV71 VP1 mRNA level and protein expression were measured using realtime fluorescence quantitative PCR and Western-blot,respectively.The GFP protein level was determined by flow cytometry,and cell viability was assessed using CCK-8 assay.The results showed that four out of the five gRNA plasmids targeting different positions of the EV71 genome significantly inhibited EV71 VP1 mRNA and protein expression,and among the four,there was one plasmid exhibiting strong collateral cleavage activity.All the plasmids showed no significant cytotoxicity.These findings suggest that the CRISPR/gRCas13d system can be used to inhibit EV71 infection,but appropriate gRNA selection is necessary to ensure antiviral efficacy and avoid cytotoxicity.

关 键 词：肠道病毒71型(EV71) 成簇规则间隔的短回文重复序列及其相关蛋白13d(CRISPR/Cas13d) 抗病毒活性 细胞毒性 

分 类 号：Q789[生物学—分子生物学] R373.2[医药卫生—病原生物学]