基于CRISPR-Cas9的痘苗病毒高效重组体系的建立

CRISPR-Cas9 system for construction of highly efficient recombinant vaccinia virus

目的利用规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)-CRISPR相关蛋白9(CRISPR associated protein 9,Cas9)技术对痘苗病毒(vaccinia virus, VACV)胸腺激酶(thymidine kinase,TK)区进行定向重组,建立高效的痘苗病毒靶基因插入重组技术。方法设计合成靶向TK区的引导RNA(guide RNA, gRNA)对应的2条互补寡核苷酸,磷酸化修饰后变性退火,克隆到去除核定位信号的PX458载体上,同时改造原有TK区重组质粒。将gRNA及Cas9共表达质粒转染293T细胞,介导VACV与TK区重组质粒进行同源重组,然后通过蓝白斑的出现率评估病毒重组率。结果本研究中设计并合成的gRNA序列介导的重组效率大于1%,高于经典的同源重组方法10倍以上。结论本研究利用CRISPR/Cas9技术建立了高效痘苗病毒TK区重组体系,为新发突发传染病的疫苗或多价研究以及肿瘤治疗等提供临床前研究技术支持。

Objective Using clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR associated protein 9(Cas9)technology to edit Vaccinia virus(VACV)thymidine kinase(TK)Region for targeted recombination to establish an efficient vaccinia virus insertion recombination technology.Methods We designed and synthesized guide RNAs(gRNA)targeting the TK region and then cloned individual gRNA into the PX458 vector that removes nuclear localization signals,and modified the original TK region recombinant plasmid.The gRNA and Cas9 co-expression plasmids were transfected into 293T cells separately to mediate the homologous recombination of vaccinia virus(VACV)and TK region recombination plasmid,and then the rate of viral recombination was evaluated by the appearance of blue and white spots.Results The recombination efficiency mediated by the gRNA sequence designed and synthesized in this study is more than 1%,which is more than 10 times higher than the classical homologous recombination method.Conclusions This study used CRISPR/Cas9 technology to establish a highly efficient recombinant vaccinia virus system,which provides technical support for pre-clinical research in vaccines or multivalent research of emerging infectious diseases,as well as tumor treatment.