基于293T-Cas9细胞株构建ICP34.5基因敲除的oHSV-1/Ecfp病毒

Constructing oHSV-1/E c f p Virus with ICP34.5 Gene Knockout Based on 293T-Cas9 Cell Line

目的构建稳定表达Cas9蛋白的工具细胞,并利用短回文重复序列/相关蛋白9(CRISPR/Cas9)编辑技术改造1型单纯疱疹病毒(HSV-1),获得ICP34.5基因敲除的oHSV-1/Ecfp重组病毒。方法利用慢病毒包装体系,将包装Cas9表达质粒的病毒感染HEK293T细胞,然后经嘌呤霉素筛选获得稳定表达Cas9蛋白的293T-Cas9细胞株,通过聚合酶链式反应(PCR)及蛋白质免疫印迹技术检测Cas9基因及蛋白表达情况;利用分子克隆技术构建HSV-1病毒ICP34.5基因敲除的基因编辑质粒(pU6-Ecfp);pU6-Ecfp转染293T-Cas9细胞后感染野生型HSV-1病毒,在胞内通过CRISPR/Cas9技术敲除HSV-1病毒中ICP34.5基因,再利用增强型青色荧光(Ecfp)示踪及极限稀释法进行病毒富集纯化,通过PCR法进行oHSV-1/Ecfp病毒鉴定。结果PCR扩增、核酸电泳及蛋白质免疫印迹技术结果显示,细胞株293T-Cas9高转录Cas9基因及高表达Cas9蛋白;通过PCR鉴定及荧光示踪结果显示,基因编辑质粒pU6-Ecfp构建成功;Ecfp荧光示踪、PCR扩增及核酸电泳结果显示,Ecfp成功插入ICP34.5基因敲除位点,获得oHSV-1/Ecfp重组病毒。结论稳定表达Cas9蛋白的293T-Cas9细胞可作为CRISPR/Cas9基因编辑的工具细胞,构建的HSV-1/Ecfp病毒实现了ICP34.5基因敲除及Ecfp示踪蛋白的敲入。

Objective To construct a stable Cas9-expressing tool cell line 293T-Cas9 and further utilize clustered regularly interspaced short palindromic repeat/CRISPR-associated protein9(CRISPR/Cas9)editing technology to modify herpes simplex virus type 1(HSV-1),aiming to obtain a recombinant virus oHSV-1/E c f p with ICP34.5 gene knockout.Methods Utilizing a lentiviral packaging system,HEK293T cells were infected with Cas9 expression plasmid-packaged virus.After puromycin selection,a stable Cas9-expressing cell line 293T-Cas9 was obtained.PCR amplification and Western Blot were used to detect the expression of Cas9.A gene-editing plasmid(pU6-E c f p)targeting the ICP34.5 gene of HSV-1 virus was constructed using molecular cloning techniques.293T-Cas9 cells were first transfected with the pU6-E c f p and then infected with wild-type HSV-1 virus.The ICP34.5 gene in HSV-1 was knocked out by CRISPR/Cas9 technology in the cell,and the virus was enriched and purified by enhanced cyan fluorescence protein(E c f p)fluorescence tracer and limit dilution method.oHSV-1/E c f p virus was identified by PCR amplification.Results PCR amplification,nucleic acid electrophoresis,and Western Blot results showed that the cell line 293T-Cas9 highly transcribed the Cas9 gene and expressed high levels of Cas9 protein.PCR amplification and fluorescent tracing results indicated that the gene editing plasmid pU6-E c f p was successfully constructed.E c f p fluorescent tracing,PCR amplification,and nucleic acid electrophoresis results revealed that E c f p was successfully inserted into the ICP34.5 gene knockout site,implying the successful construction of the recombinant virus oHSV-1/E c f p.Conclusion The stable Cas9-expressing cell 293T-Cas9 can be used as a tool cell for CRISPR/Cas9 gene editing.The constructed HSV-1/E c f p virus achieved ICP34.5 gene knockout and E c f p tracer protein knock-in.