Virology
Construction of recombinant herpes simplex virus 2 expressing enhanced green fluorescent protein using CRISPR/Cas9
Su Wenhao, Ren Xiuxiu, Zhao Tingting, Wang Yinan, Li Shishi, Huang Qiufang, Wang Xiaojie, Zhang Xiaohuan, Wei Jiangbo
Published 2022-05-31
Cite as Chin J Microbiol Immunol, 2022, 42(5): 369-375. DOI: 10.3760/cma.j.cn112309-20211021-00348
Abstract
ObjectiveTo 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.
MethodsFour 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.
ResultsThe 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.
ConclusionsLinearized 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.
Key words:
CRISPR/Cas9; Herpes simplex virus 2; Nonhomologous end-joining; Homology-directed repair
Contributor Information
Su Wenhao
Laboratory of Herpes Virus, National Vaccine and Serum Institute, Beijing 101111, China
Ren Xiuxiu
Laboratory of Herpes Virus, National Vaccine and Serum Institute, Beijing 101111, China
Zhao Tingting
Laboratory of Herpes Virus, National Vaccine and Serum Institute, Beijing 101111, China
Wang Yinan
Laboratory of Herpes Virus, National Vaccine and Serum Institute, Beijing 101111, China
Li Shishi
Laboratory of Herpes Virus, National Vaccine and Serum Institute, Beijing 101111, China
Huang Qiufang
Laboratory of Herpes Virus, National Vaccine and Serum Institute, Beijing 101111, China
Wang Xiaojie
Laboratory of Herpes Virus, National Vaccine and Serum Institute, Beijing 101111, China
Zhang Xiaohuan
Laboratory of Herpes Virus, National Vaccine and Serum Institute, Beijing 101111, China
Wei Jiangbo
Laboratory of Herpes Virus, National Vaccine and Serum Institute, Beijing 101111, China
