爲快速、高效構建僞狂犬病病毒（p地做seudorabies virus，PRV）糖蛋白路街E基因（gE）缺失病毒，基于CRI討如SPR/Cas9基因編輯技術，首先將(ji年得āng)pSpCas9（BB）-2A-GFP熒光質粒轉染至友家VERO細胞和PK-15細胞，選出轉染效率較高的細胞系，同時(shí)于ht秒自tp://crispr.mit.edu拿這/網站設計并合成(chéng)3個高評分的小導向(xiàng)RN兒信A（small guide RN章近A，sgRNA），通過(guò)噬斑形成(chéng)試業錢驗，篩選出高效sgRNA。其次低照將(jiāng)篩選出的針對(duì)PR員資V gE基因的sgRNA轉染于PK-1錯輛5細胞，然後(hòu)接種(zhǒng)PRV-1病毒，經(j能員īng)過(guò)5 輪噬斑克隆純化獲得PRV-1-ΔgE。結服志果顯示：VERO細胞比PK-15細胞具有更好(電店hǎo)的轉染效果；gE-sgRNA1和gE-sg飛子RNA2可作爲針對(duì)gE基因的高效sgRNA；獲得了1株雜跳PRV gE基因缺失291 bp的病毒，將(體著jiāng)其命名爲PRV-1-ΔgE。研究表明，CRISPR/Cas師呢9基因編輯技術可作爲一種(zh裡煙ǒng)高效編輯PRV-1缺失病毒基因的方法，同時(shí)也爲後(hòu)續務樂快速應對(duì)PRV變異株研究提供了新思路。
Construction of姐關 PRV gE-deleted Strain Based 農美on CRISPR/Cas9 不站Technology
In order to rapidl湖小y and effectively construct the gE用睡-deleted strain of pseudo體哥rabies virus（PRV），the fluorescent 兵服plasmid of pSpCas9（BB）-2A-GFP was first師路ly transfected i姐到nto VERO cells and PK-15 cells to sele畫亮ct the cell lines with hig們美h efficiency. Meanwhile，three small 光知guide RNAs（sgRNAs）with hi我林gh scores were design說下ed and synthesi火金zed on http://crispr.習年mit.edu/ to select high-efficiency 亮車sgRNAs through plaque formation ex著生periments. Then the 家如selected sgRNA w身低as transfected int鐘師o PK-15 cells which subseq技小uently were inoculated with PRV車是-1，and finally PRV-1-爸知ΔgE was obtained aft民金er five rounds of plaque clon唱間e purification. The results showed th服錢at VERO cells could be we又也ll transfected compared to PK-街你15 cells；gE-sgRNA1開月 and gE-sgRNA2 could be used as生電 the high-efficiency sgRNAs for議跳 gE gene；a strain of gE-deleted viru亮作s with a deletion of 291 b快通p was obtained an亮裡d named as PRV-1-ΔgE. It was found th輛媽at CRISPR/Cas9 gene editing techn靜黑ology could be used for effective要冷ly editing PRV-1 gene-dele黃窗ted strain，which als讀資o provided new ideas to rapidly r秒說esponse to PRV varian劇科ts in the future.
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