基于CRISPR/Cas9技术快速构建PRV gE基因缺失病毒

Construction of PRV gE-deleted Strain Based on CRISPR/Cas9 Technology

为快速、高效构建伪狂犬病病毒(pseudorabies virus,PRV)糖蛋白E基因(gE)缺失病毒,基于CRISPR/Cas9基因编辑技术,首先将pSpCas9(BB)-2A-GFP荧光质粒转染至VERO细胞和PK-15细胞,选出转染效率较高的细胞系,同时于http://crispr.mit.edu/网站设计并合成3个高评分的小导向RNA(small guide RNA,sgRNA),通过噬斑形成试验,筛选出高效sgRNA。其次将筛选出的针对PRV gE基因的sgRNA转染于PK-15细胞,然后接种PRV-1病毒,经过5轮噬斑克隆纯化获得PRV-1-ΔgE。结果显示:VERO细胞比PK-15细胞具有更好的转染效果;gE-sgRNA1和gE-sgRNA2可作为针对gE基因的高效sgRNA;获得了1株PRV gE基因缺失291 bp的病毒,将其命名为PRV-1-ΔgE。研究表明,CRISPR/Cas9基因编辑技术可作为一种高效编辑PRV-1缺失病毒基因的方法,同时也为后续快速应对PRV变异株研究提供了新思路。

In order to rapidly and effectively construct the gE-deleted strain of pseudorabies virus(PRV),the fluorescent plasmid of pSpCas9(BB)-2 A-GFP was firstly transfected into VERO cells and PK-15 cells to select the cell lines with high efficiency. Meanwhile,three small guide RNAs(sgRNAs)with high scores were designed and synthesized on http://crispr.mit.edu/to select high-efficiency sgRNAs through plaque formation experiments. Then the selected sgRNA was transfected into PK-15 cells which subsequently were inoculated with PRV-1,and finally PRV-1-ΔgE was obtained after five rounds of plaque clone purification. The results showed that VERO cells could be well transfected compared to PK-15 cells;gE-sgRNA1 and gE-sgRNA2 could be used as the high-efficiency sgRNAs for gE gene;a strain of gE-deleted virus with a deletion of 291 bp was obtained and named as PRV-1-ΔgE. It was found that CRISPR/Cas9 gene editing technology could be used for effectively editing PRV-1 gene-deleted strain,which also provided new ideas to rapidly response to PRV variants in the future.