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Cytoplasmic-replicating viruses use their own repair enzymes for homologous recombination, while nuclear-replicating viruses use the host repair machinery. This is translated into a wide range of Cas9-induced homologous recombination efficiencies, depending on the virus replication compartment and viral\/host repair machinery characteristics and accessibility. However, the use of Cas9 as a selection agent to target parental virus genomes robustly improves the selection of desired recombinants across large dsDNA viruses. We used ectromelia virus (ECTV) and herpes simplex virus (HSV) type 1 and 2 to optimize a CRISPR\/Cas9 method that can be used versatilely for efficient genome editing and selection of both cytoplasmic- and nuclear-replicating viruses. We performed a genome-wide genetic variant analysis of mutations located at predicted off-target sequences for 20 different recombinants, showing off-target-free accuracy by deep sequencing. Our results support this optimized method as an efficient, accurate and versatile approach to enhance the two critical factors of high-throughput viral genome engineering: generation and colour-based selection of recombinants. This application of CRISPR\/Cas9 reduces the time and labour for screening of desired recombinants, allowing for high-throughput generation of large collections of mutant dsDNA viruses for a desired locus, optimally in less than 2\u2009weeks.<\/jats:p>","DOI":"10.1099\/jgv.0.001797","type":"journal-article","created":{"date-parts":[[2022,10,19]],"date-time":"2022-10-19T10:18:05Z","timestamp":1666174685000},"source":"Crossref","is-referenced-by-count":3,"title":["High-throughput engineering of cytoplasmic- and nuclear-replicating large dsDNA viruses by CRISPR\/Cas9"],"prefix":"10.1099","volume":"103","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1542-2153","authenticated-orcid":false,"given":"Alberto Domingo","family":"L\u00f3pez-Mu\u00f1oz","sequence":"first","affiliation":[{"name":"Present address: Cellular Biology Section, Laboratory of Viral Diseases, NIAID, NIH, Bethesda, Maryland, USA"},{"name":"Centro de Biolog\u00eda Molecular Severo Ochoa, Consejo Superior de 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