Phages are important drivers of bacterial evolution and have potential as antimicrobials. However, incomplete understanding of phage biology and our inability to rapidly engineer them with new genetic cargo hinder progress towards phage-based therapies. Here we develop an unbiased, genome-wide mutational tool for phages. This approach, phage Tn-seq, uses Tn5 transposon mutagenesis using anti-CRISPR-based selection and deep-sequencing. Phage Tn-seq was successful across diverse phages, including a nucleus-forming jumbo phage and enabled gene essentiality assignment consistent with phage structural proteomics and core gene conservation. In addition, insertion biases allowed prediction of transcriptional direction and early injected and highly expressed regions. We exploit transposons to deliver new cargo to phages within a few days and created an orthogonal artificial intelligence-designed Acr transposon system to generate phage double mutants. Transposon insertion was also achieved in phages with hypermodified DNA. Phage Tn-seq is a versatile tool to advance our understanding and application of phages. Phage Tn-seq uses Tn5 transposon mutagenesis with anti-CRISPR-based selection and deep sequencing as a method to generate unbiased, genome-wide mutations across diverse phages
