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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.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.02048-25","type":"journal-article","created":{"date-parts":[[2026,2,18]],"date-time":"2026-02-18T14:01:53Z","timestamp":1771423313000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":3,"title":["Viral genome editing methods and applications in the CRISPR era"],"prefix":"10.1128","volume":"100","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8044-4530","authenticated-orcid":true,"given":"Kihye","family":"Shin","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, Jeju National University College of Medicine","place":["Jeju, Republic of 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