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Even in well-characterized phages, the functions of most genes remain unknown. Experimental approaches to study phage gene fitness and function at genome scale are lacking, partly because phages subvert many modern functional genomics tools. Here we leverage RNA-targeting dCas13d to selectively interfere with protein translation and to measure phage gene fitness at a transcriptome-wide scale. We find CRISPR Interference through Antisense RNA-Targeting (CRISPRi-ART) to be effective across phage phylogeny, from model ssRNA, ssDNA and dsDNA phages to nucleus-forming jumbo phages. Using CRISPRi-ART, we determine a conserved role of diverse rII homologues in subverting phage Lambda RexAB-mediated immunity to superinfection and identify genes critical for phage fitness. CRISPRi-ART establishes a broad-spectrum phage functional genomics platform, revealing more than 90 previously unknown genes important for phage fitness.<\/jats:p>","DOI":"10.1038\/s41564-025-01935-7","type":"journal-article","created":{"date-parts":[[2025,2,26]],"date-time":"2025-02-26T10:03:45Z","timestamp":1740564225000},"page":"694-709","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":32,"title":["CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d"],"prefix":"10.1038","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7488-3040","authenticated-orcid":false,"given":"Benjamin A.","family":"Adler","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muntathar J.","family":"Al-Shimary","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jaymin R.","family":"Patel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0377-1209","authenticated-orcid":false,"given":"Emily G.","family":"Armbruster","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Colognori","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emeric J.","family":"Charles","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5315-4172","authenticated-orcid":false,"given":"Kate V.","family":"Miller","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arushi","family":"Lahiri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael L.","family":"Cui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Agn\u00e8s","family":"Orom\u00ed-Bosch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angela","family":"Voelker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7839-4642","authenticated-orcid":false,"given":"Marena","family":"Trinidad","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jina","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sebastien","family":"Beurnier","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4467-271X","authenticated-orcid":false,"given":"Ron","family":"Boger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jason","family":"Nomburg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0648-3504","authenticated-orcid":false,"given":"Rodolphe","family":"Barrangou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7934-0400","authenticated-orcid":false,"given":"Vivek K.","family":"Mutalik","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph S.","family":"Schoeniger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8989-2655","authenticated-orcid":false,"given":"Joseph A.","family":"Pogliano","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0042-2257","authenticated-orcid":false,"given":"David F.","family":"Savage","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9161-999X","authenticated-orcid":false,"given":"Jennifer A.","family":"Doudna","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2948-2846","authenticated-orcid":false,"given":"Brady F.","family":"Cress","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,2,26]]},"reference":[{"key":"1935_CR1","doi-asserted-by":"publisher","first-page":"31","DOI":"10.4161\/bact.1.1.14942","volume":"1","author":"MR Clokie","year":"2011","unstructured":"Clokie, M. 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J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Evercrisp and Azalea; a scientific advisory board member of Caribou Biosciences, Scribe Therapeutics, Mammoth and Inari; and a Director at Johnson & Johnson, Tempus and Altos Labs. R. Barrangou. is a shareholder of Caribou Biosciences, Intellia Therapeutics, Locus Biosciences, Inari, TreeCo, and Ancilia Biosciences. The other authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}]}}