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Here, we applied this approach to Kaposi\u2019s sarcoma-associated herpesvirus (KSHV) by designing a sgRNA library containing all possible ~22,000 guides targeting the 154 kilobase viral genome, corresponding to one cut site approximately every 8 base pairs. We used the library to profile viral sequences involved in transcriptional activation of late genes, whose regulation involves several well characterized features including dependence on viral DNA replication and a known set of viral transcriptional activators. Upon phenotyping all possible Cas9-targeted viruses for transcription of KSHV late genes we recovered these established regulators and identified a new required factor (ORF46), highlighting the utility of the screening pipeline. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we identify the DNA binding but not catalytic domain of ORF46 to be required for viral DNA replication and thus late gene expression. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.<\/jats:p>","DOI":"10.1371\/journal.ppat.1010236","type":"journal-article","created":{"date-parts":[[2022,1,18]],"date-time":"2022-01-18T15:22:30Z","timestamp":1642519350000},"page":"e1010236","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":11,"title":["A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains"],"prefix":"10.1371","volume":"18","author":[{"given":"David W.","family":"Morgens","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Divya","family":"Nandakumar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7277-5233","authenticated-orcid":true,"given":"Allison L.","family":"Didychuk","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kevin J.","family":"Yang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0479-9377","authenticated-orcid":true,"given":"Britt A.","family":"Glaunsinger","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2022,1,18]]},"reference":[{"key":"ppat.1010236.ref001","doi-asserted-by":"crossref","first-page":"320","DOI":"10.1038\/s41588-019-0558-9","article-title":"The landscape of viral associations in human cancers","volume":"52","author":"M Zapatka","year":"2020","journal-title":"Nat Genet"},{"key":"ppat.1010236.ref002","doi-asserted-by":"crossref","first-page":"803","DOI":"10.1158\/1078-0432.CCR-0670-3","article-title":"Epstein-Barr Virus and Cancer.","author":"MP Thompson","year":"2004","journal-title":"Clinical Cancer Research"},{"key":"ppat.1010236.ref003","doi-asserted-by":"crossref","first-page":"939","DOI":"10.1172\/JCI40567","article-title":"KSHV and the pathogenesis of Kaposi sarcoma: listening to human biology and medicine","volume":"120","author":"D. 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