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Despite their importance in bacterial ecology and evolutionary processes, phage genes are poorly characterized, hampering their usage in a variety of biotechnological applications. Methods to characterize such genes, even those critical to the phage life cycle, are labor intensive and are generally phage specific. Here, we develop a systematic gene essentiality mapping method scalable to new phage\u2013host combinations that facilitate the identification of nonessential genes. As a proof of concept, we use an arrayed genome-wide CRISPR interference (CRISPRi) assay to map gene essentiality landscape in the canonical coliphages \u03bb and P1. Results from a single panel of CRISPRi probes largely recapitulate the essential gene roster determined from decades of genetic analysis for lambda and provide new insights into essential and nonessential loci in P1. We present evidence of how CRISPRi polarity can lead to false positive gene essentiality assignments and recommend caution towards interpreting CRISPRi data on gene essentiality when applied to less studied phages. Finally, we show that we can engineer phages by inserting DNA barcodes into newly identified inessential regions, which will empower processes of identification, quantification, and tracking of phages in diverse applications.<\/jats:p>","DOI":"10.1371\/journal.pbio.3002416","type":"journal-article","created":{"date-parts":[[2023,12,4]],"date-time":"2023-12-04T18:29:06Z","timestamp":1701714546000},"page":"e3002416","update-policy":"https:\/\/doi.org\/10.1371\/journal.pbio.corrections_policy","source":"Crossref","is-referenced-by-count":34,"title":["Systematic and scalable genome-wide essentiality mapping to identify nonessential genes in phages"],"prefix":"10.1371","volume":"21","author":[{"given":"Denish","family":"Piya","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas","family":"Nolan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Madeline 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