Viral genome editing methods and applications in the CRISPR era.

CRISPR-Cas systems have transformed viral genetics by enabling precise and efficient manipulation of large DNA virus genomes. This review provides a practical framework for applying CRISPR technology to herpesviruses and other large DNA viruses as an alternative and complement to traditional BAC recombination. Key considerations include nuclease choice; sgRNA design that minimizes cut-to-edit distance and prevents re-cutting; donor template configuration and homology arm length; and synchronized delivery of Cas complexes and donor DNA. Strategies to promote HDR efficiency, such as the use of small-molecule modulators, are also summarized. In addition, practical workflows for clone selection, genotypic validation, and phenotypic confirmation are summarized. Case studies in herpes simplex virus type 1 and human cytomegalovirus illustrate how optimized CRISPR designs achieve reproducible, scarless knock-ins and conditional gene manipulation at essential loci without complementing cell lines. Together, these approaches establish CRISPR as a flexible, scalable platform for functional genomics, antiviral target discovery, and translational virology, enabling direct editing of clinical isolates previously inaccessible with bacterial artificial chromosome-based methods.