Liu Chun-Yu, Jin Ming, Guo Hao, Zhao Hong-Zhe, Hou Li-Na, Yang Yang, Wen Yong-Jun, Wang Feng-Xue
College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China.
School of Life Sciences, Inner Mongolia University, Hohhot 010018, China.
Vet Sci. 2022 Mar 30;9(4):166. doi: 10.3390/vetsci9040166.
Bovine herpesvirus type I (BoHV-1) is an important pathogen that causes respiratory disease in bovines. The disease is prevalent worldwide, causing huge economic losses to the cattle industry. Gene-deficient vaccines with immunological markers to distinguish them from wild-type infections have become a mainstream in vaccine research and development. In order to knock out the gE gene BoHV-1, we employed the CRISPR/Cas9 system. Interesting phenomena were observed at the single guide RNA (sgRNA) splicing site, including gene insertion, gene deletion, and the inversion of 5' and 3' ends of the sgRNA splicing site. In addition to the deletion of the gE gene, the US9 gene, and the non-coding regions of gE and US9, it was found that the US4 sequence, US6 sequence, and part of the US7 sequence were inserted into the EGFP sgRNA splicing site and the 3' end of the EGFP sequence was deleted. Similar to the BoHV-1 parent, the BoHV-1 mutants induced high neutralizing antibodies titer levels in mice. In summary, we developed a series of recombinant gE-deletion BoHV-1 samples using the CRISPR/Cas9 gene editing system. The mutant viruses with EGFP or EGFP will lay the foundation for research on BoHV-1 and vaccine development in the future.
牛I型疱疹病毒（BoHV-1）是一种导致牛呼吸道疾病的重要病原体。该疾病在全球范围内流行，给养牛业造成巨大经济损失。带有免疫标记以区分野生型感染的基因缺陷疫苗已成为疫苗研发的主流。为了敲除BoHV-1的gE基因，我们采用了CRISPR/Cas9系统。在单导向RNA（sgRNA）剪接位点观察到有趣的现象，包括基因插入、基因缺失以及sgRNA剪接位点5'和3'末端的倒置。除了gE基因、US9基因以及gE和US9的非编码区缺失外，还发现US4序列、US6序列和部分US7序列插入到EGFP sgRNA剪接位点，且EGFP序列的3'末端缺失。与BoHV-1亲本相似，BoHV-1突变体在小鼠体内诱导出高中和抗体滴度水平。总之，我们利用CRISPR/Cas9基因编辑系统开发了一系列重组gE缺失的BoHV-1样本。带有EGFP或EGFP的突变病毒将为未来BoHV-1研究和疫苗开发奠定基础。
