Development of a Markerless Gene Deletion System for HSV-1

HSV-1 is a widespread human pathogen that causes various diseases worldwide, highlighting the need for novel approaches to treatment and prevention. However, the lack of some efficient gene deletion techniques has hampered the pathological studies, pharmaceutical research and vaccines development of this virus. In this study, we developed an efficient and markerless deletion system mainly composed of two recombinant plasmids, BAC-HSV-1 and pREDI. And the homologous fragment a-c-Kan-sacB-b-c used in this system contained three homology regions and two selection markers (positive/ counter). Rapid deletion was achieved in three steps (1) amplification of homologous fragment, (2) antibiotic and sucrose screening of recombinants, and (3) BAC excision. The overall deletion efficiency was greater than 83.9 %. We were the first to develop a special in-vitro gene deletion system to aim to delete HSV-1 genes. And we had successfully deleted the ICP34.5, UL5, UL41, UL29, and US8 genes of HSV-1 and conducted preliminary analysis of the virus titer of mutants.