基于CRISPR/Cas9技术的重组病毒HSV-2-EGFP的构建

Construction of recombinant herpes simplex virus 2 expressing enhanced green fluorescent protein using CRISPR/Cas9

目的利用CRISPR/Cas9(clustered,regularly interspaced,short palindromic repeat/CRISPR-associated nuclease 9)技术构建携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因的单纯疱疹病毒2型(herpes simplex virus 2,HSV-2)。方法利用CRISPR/Cas9技术对外源基因EGFP插入HSV-2基因组的策略进行探索,设计如下4种插入策略:(1)经典的同源重组修复模式,即环状双侧同源臂供体介导的基因敲入;(2)线性化单侧同源臂供体介导的基因敲入;(3)同源性非依赖介导的基因敲入;(4)利用稳表达Cas9和sgRNA的细胞株,进行环状双侧同源臂供体介导的基因敲入。结果使用策略2、3和4均成功构建了携带EGFP的HSV-2,其中策略2的基因敲入效率最高,然后依次为策略3、策略4,策略1未观察到重组病毒的产生。蚀斑纯化后的重组病毒在7代内能稳定表达绿色荧光蛋白,并且和亲本株在Vero细胞上具有相似的生长特性。结论线性化单侧同源臂供体介导的基因敲入效率更高,敲除载体的稳转细胞株能够有效地提高同源重组修复机制介导的基因敲入效率。

Objective To construct a recombinant herpes simplex virus 2(HSV-2)expressing enhanced green fluorescent protein(EGFP)using clustered,regularly interspaced,short palindromic repeat/CRISPR-associated nuclease 9(CRISPR/Cas9)technology.Methods Four strategies for inserting exogenous EGFP gene into HSV-2 genome using CRISPR/Cas9 technology were designed:(1)conventional homology-directed repair:circular two homology arm donor-mediated gene knock-in;(2)linearized single homology arm donor-mediated gene knock-in;(3)homology-independent targeted integration;(4)conventional homology-directed repair-mediated by cell lines stably expressing Cas9 and sgRNA.Results The recombinant virus HSV-2-EGFP was successfully constructed based on the second,the third and the fourth strategies.The second strategy was the most efficient,followed by the third and the fourth strategies.The purified recombinant virus could stably express green fluorescent protein in seven passages and shared similar growth characteristics in Vero cells to the parental virus.Conclusions Linearized single homology arm donor could increase the efficiency of gene knock-in,and cell lines stably expressing Cas9 and sgRNA could increase the efficiency of gene knock-in mediated by homology-directed repair.