Rapid construction of rPRV-ΔTK/EGFP variant strain using CRISPR/Cas9 sys-tem
10.16303/j.cnki.1005-4545.2024.06.17
- VernacularTitle:利用CRISPR/Cas9系统快速构建携带EGFP的PRV-ΔTK重组变异株
- Author:
Zaijiao YE
1
,
2
,
3
;
Chuan ZENG
;
Jun GU
;
Peixia WANG
;
Jinyan SHEN
;
Deping SONG
;
Dongyan HUANG
;
Xiangdong WU
;
Houjun HE
;
Yuxin TANG
;
Yu YE
Author Information
1. 江西农业大学 动物科学技术学院,江西 南昌 330045
2. 江西省动物疫病防控制剂工程研究中心,江西 南昌 330045
3. 江西农业大学 工学院,江西 南昌 330045
- Keywords:
PRV;
CRISPR/Cas9;
EGFP;
recombination;
plaque purification
- From:
Chinese Journal of Veterinary Science
2024;44(6):1223-1228
- CountryChina
- Language:Chinese
-
Abstract:
Pseudorabies virus(PRV)is the etiological agent of pseudorabies in pigs,which is char-acterized by dyspnea,reproductive disorders,and neurological diseases,and it spreads widely a-round the world.Since 2011,the newly emerged PRV variants have resulted in poor immunity pro-tection of traditional vaccine strains,and the original method of vaccine strain preparation is time-consuming and labor-intensive.Therefore,it is urgently needed to develop an efficient screening method of the vaccine strain at present.Using CRISPR/Cas9 gene editing technology in this study,two single guide RNAs(sgRNA)were designed targeting the virulence gene TK of PRV variant strain CH/JX/2016,and then the enhanced green fluorescent protein the reporter(EGFP)gene was inserted at the TK locus by a homologous repair plasmid.After multiple rounds of plaque puri-fication,the rPRV-ΔTK/EGFP strain was obtained.The results showed the cleavage efficiency of the two sgRNAs was extremely high.The preparation of rPRV-ΔTK/EGFP strain was succeed af-ter only three rounds of purification,and the EGFP expressed normally.The CRISPR/Cas9 system can edit the PRV gene simply,rapidly,and efficiently,and exhibits great potential in the construction of vaccine candidate strains.Meanwhile,the rescued rPRV-ΔTK/EGFP strain not only could be used as a tracer strain in PRV variant infection progresses,but also for subsequent antivi-ral drug screening.
