CRISPR/Cas9-Mediated Point Mutation in Essential HSV-1 Genes Using an IRES-Based Strategy

Herpes simplex virus type 1 (HSV-1) is a large, double-stranded DNA virus whose genome encodes numerous essential genes required for viral replication and pathogenesis.Genetic manipulation of these essential genes remains challenging because conventional CRISPR/Cas9-based editing strategies often involve initial deletion of the target gene, resulting in loss of viral viability.To overcome this limitation, we developed a CRISPR/Cas9-based GFP cassette insertion-replacement strategy incorporating an internal ribosome entry site (IRES), enabling bicistronic expression of a fluorescent selection marker and an essential viral gene without disrupting gene function.As proof of concept, we targeted the essential tegument gene UL36 and inserted a CMV promoter-eGFP-IRES cassette upstream of its ORF, allowing simultaneous expression of eGFP and UL36.The cassette was subsequently replaced with a UL36 mutant harboring a deubiquitinase (DUB)-inactive C40S substitution.This approach preserved viral viability and achieved high homology-directed repair (HDR) efficiency, allowing precise introduction of point mutations into essential genes.Our IRES-guided genome editing strategy provides a robust and versatile platform for precise manipulation of essential genes in HSV-1 and other large DNA viruses.