Concurrent Gene Insertion, Deletion, and Inversion during the Construction of a Novel Attenuated BoHV-1 Using...

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.