Construction of recombinant herpes simplex virus 2 expressing enhanced green fluorescent protein using CRISPR/Cas9
10.3760/cma.j.cn112309-20211021-00348
- VernacularTitle:基于CRISPR/Cas9技术的重组病毒HSV-2-EGFP的构建
- Author:
Wenhao SU
1
;
Xiuxiu REN
;
Tingting ZHAO
;
Yinan WANG
;
Shishi LI
;
Qiufang HUANG
;
Xiaojie WANG
;
Xiaohuan ZHANG
;
Jiangbo WEI
Author Information
1. 国药中生生物技术研究院疱疹病毒实验室，北京 101111
- Keywords:
CRISPR/Cas9;
Herpes simplex virus 2;
Nonhomologous end-joining;
Homology-directed repair
- From:
Chinese Journal of Microbiology and Immunology
2022;42(5):369-375
- CountryChina
- Language:Chinese
-
Abstract:
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.
