利用CRISPR/Cas9系统快速构建携带EGFP的PRV-ΔTK重组变异株

Rapid construction of rPRV-ΔTK/EGFP variant strain using CRISPR/Cas9 system

伪狂犬病病毒(pseudorabies virus,PRV)能够引起猪只出现以呼吸困难、繁殖障碍和神经系统疾病等为特征的伪狂犬病,在世界范围内传播广泛。2011年以来,新现的PRV变异株导致传统疫苗株的免疫保护效果不佳,且原有的疫苗株制备方法耗时耗力,因此现阶段急需开发一种高效疫苗株筛选的方法。采用CRISPR/Cas9基因编辑技术,设计2条靶向PRV毒力基因TK的单链引导RNA(single guide RNA,sgRNA),敲除PRV变异株CH/JX/2016编码的TK基因,再利用同源修复质粒在TK基因座位置插入增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP),经多轮蚀斑纯化获得rPRV-ΔTK/EGFP毒株。结果表明本研究构建的sgRNA切割效率高,只经过3轮纯化就可完成rPRV-ΔTK/EGFP毒株的制备,且EGFP基因正常表达。CRISPR/Cas9系统能够简便快速高效地编辑PRV基因,在疫苗候选株创制中潜力巨大,而且拯救的rPRV-ΔTK/EGFP毒株不仅可以作为研究PRV变异株感染进程的示踪毒株,还能用于后续抗病毒药物的筛选。

Pseudorabies virus(PRV)is the etiological agent of pseudorabies in pigs,which is characterized by dyspnea,reproductive disorders,and neurological diseases,and it spreads widely around the world.Since 2011,the newly emerged PRV variants have resulted in poor immunity protection of traditional vaccine strains,and the original method of vaccine strain preparation is timeconsuming and labor-intensive.Therefore,it is urgently needed to develop an efficient screening method of the vaccine strain at present.Using CRISPR/Cas9 gene editing technology in this study,two single guide RNAs(sgRNA)were designed targeting the virulence gene TK of PRV variant strain CH/JX/2016,and then the enhanced green fluorescent protein the reporter(EGFP)gene was inserted at the TK locus by a homologous repair plasmid.After multiple rounds of plaque purification,the rPRV-ΔTK/EGFP strain was obtained.The results showed the cleavage efficiency of the two sgRNAs was extremely high.The preparation of rPRV-ΔTK/EGFP strain was succeed after only three rounds of purification,and the EGFP expressed normally.The CRISPR/Cas9system can edit the PRV gene simply,rapidly,and efficiently,and exhibits great potential in the construction of vaccine candidate strains.Meanwhile,the rescued rPRV-ΔTK/EGFP strain not only could be used as a tracer strain in PRV variant infection progresses,but also for subsequent antiviral drug screening.