CRISPR/Cas9切割痘病毒DNA抑制病毒复制  

CRISPR/Cas9 Cleavage of Viral DNA Efficiently Suppresses Vaccinia Viruses

作　　者：王娇娇[1] 张新敏[1] 倪爱民[2] 章康健[2] 刘新垣[1,2] 刘锡君[3] 

机构地区：[1]浙江理工大学生命科学学院新元医学与生物技术研究所,杭州310018 [2]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200031 [3]同济大学附属第十人民医院泛血管疾病研究所,上海200072

出　　处：《中国细胞生物学学报》2016年第4期405-412,共8页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:31301064);同济大学青年优秀人才培养行动计划(批准号:2015KJ056)资助的课题~~

摘　　要：当前,CRISPR/Cas9系统靶向痘苗病毒的相关研究鲜有报道。该文采用了CRISPR/Cas9基因组定点编辑技术,以痘病毒为研究对象。在重组痘病毒WR-EGFP中针对EGFP基因序列设计不同靶点g RNAs,转染Cas9/g RNA质粒,同时感染WR-EGFP痘病毒,随后观察EGFP荧光表达、提取病毒基因组,通过PCR进行切口鉴定并运用错配酶切法验证CRISPR/Cas9对痘病毒靶点DNA切割的切口修复情况。与单独感染WR-EGFP组相比,转染了Cas9/g RNA174和Cas9/g RNA175质粒的EGFP荧光强度明显降低,表明CRISPR/Cas9抑制WR-EGFP病毒中EGFP基因的表达。相对定量PCR及错配酶切实验结果显示,CRISPR/Cas9切割靶点导致WR-EGFP病毒基因组产生了切口,并且存在DNA错配修复现象。最后,通过结晶紫染色法测定痘病毒的滴度,结果显示,经CRISPR/Cas9处理组的痘病毒滴度显著下降,即CRISPR/Cas9能够使痘病毒的复制能力显著地降低。综上所述,CRISPR/Cas9切割痘病毒基因组DNA并且抑制痘病毒复制。Previous study had shown that viral genomes could be edited effectively using CRISPR/Cas9-mediated genomic editing technique. However, the precise edit to viruses with large DNA genomes, such as vaccinia viruses by CRISPR/Cas9 system has not been reported. To explore the effects of genetic inactivation or mutation on vaccinia viral replication, the recombinant virus armed with an nonessential enhanced green fluorescent protein（EGFP） gene（WR-EGFP） was selected as the object of study here. Two guide RNAs were designed to target the coding region of EGFP gene. Fluorescence microscopy result showed that expression of EGFP significantly decreased in cells that both infected with WR-EGFP viruses and transfected with Cas9/g RNA plasmids, compared with control group that only infected with WR-EGFP. The Cas9/g RNA could induce site-specific cleavage of EGFP gene, thus caused mismatch repair of DNA viral genomes which were detected by SURVEYOR assay. Moreover, the mismatch repair of DNA sequence in virus resulted in losing the ability of virus replication. Taken together, the CRISPR/Cas9 system was a highly efficient strategy for editing the recombinant vaccinia viral genomes which could significantly inhibit the replication of virus.

关 键 词：CRISPR/Cas9 痘病毒 靶向切割 病毒复制 

分 类 号：Q78[生物学—分子生物学]