CRISPR-Cas9 system for construction of highly efficient recombinant vaccinia virus
10.3760/cma.j.cn112866-20210111-00008
- VernacularTitle:基于CRISPR-Cas9的痘苗病毒高效重组体系的建立
- Author:
Yabin WU
1
;
Li ZHAO
;
Jiao REN
;
Hang YUAN
;
Peng ZHANG
;
Fei YE
;
Houwen TIAN
;
Wenling WANG
;
Wenjie TAN
Author Information
1. 中国疾病预防控制中心病毒病预防控制所，北京 102206
- Keywords:
Vaccinia virus;
CRISPR/Cas9;
Efficient recombination system
- From:
Chinese Journal of Experimental and Clinical Virology
2021;35(2):199-204
- CountryChina
- Language:Chinese
-
Abstract:
Objective:Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) technology to edit Vaccinia virus (VACV) thymidine kinase (TK) Region for targeted recombination to establish an efficient vaccinia virus insertion recombination technology.Methods:We designed and synthesized guide RNAs(gRNA)targeting the TK region and then cloned individual gRNA into the PX458 vector that removes nuclear localization signals, and modified the original TK region recombinant plasmid. The gRNA and Cas9 co-expression plasmids were transfected into 293T cells separately to mediate the homologous recombination of vaccinia virus (VACV) and TK region recombination plasmid, and then the rate of viral recombination was evaluated by the appearance of blue and white spots.Results:The recombination efficiency mediated by the gRNA sequence designed and synthesized in this study is more than 1%, which is more than 10 times higher than the classical homologous recombination method .Conclusions:This study used CRISPR/Cas9 technology to establish a highly efficient recombinant vaccinia virus system, which provides technical support for pre-clinical research in vaccines or multivalent research of emerging infectious diseases, as well as tumor treatment.
