為快速、高效構(gòu)建偽狂犬病病毒（pseudorabies virus，PRV）糖蛋白E基因（gE）缺失病毒，基于CRISPR/Cas9基因編輯技術(shù)，首先將pSpCas9（BB）-2A-GFP熒光質(zhì)粒轉(zhuǎn)染至VERO細(xì)胞和PK-15細(xì)胞，選出轉(zhuǎn)染效率較高的細(xì)胞系，同時(shí)于http://crispr.mit.edu/網(wǎng)站設(shè)計(jì)并合成3個(gè)高評(píng)分的小導(dǎo)向RNA（small guide RNA，sgRNA），通過(guò)噬斑形成試驗(yàn)，篩選出高效sgRNA。其次將篩選出的針對(duì)PRV gE基因的sgRNA轉(zhuǎn)染于PK-15細(xì)胞，然后接種PRV-1病毒，經(jīng)過(guò)5 輪噬斑克隆純化獲得PRV-1-ΔgE。結(jié)果顯示：VERO細(xì)胞比PK-15細(xì)胞具有更好的轉(zhuǎn)染效果；gE-sgRNA1和gE-sgRNA2可作為針對(duì)gE基因的高效sgRNA；獲得了1株P(guān)RV gE基因缺失291 bp的病毒，將其命名為PRV-1-ΔgE。研究表明，CRISPR/Cas9基因編輯技術(shù)可作為一種高效編輯PRV-1缺失病毒基因的方法，同時(shí)也為后續(xù)快速應(yīng)對(duì)PRV變異株研究提供了新思路。
Construction of PRV gE-deleted Strain Based on CRISPR/Cas9 Technology
In order to rapidly and effectively construct the gE-deleted strain of pseudorabies virus（PRV），the fluorescent plasmid of pSpCas9（BB）-2A-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.
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