Tn5-EGIM: efficient Tn5 transposon mutagenesis to identify non-essential bacteriophage genes for genome minimization and engineering

Abstract Generating high‑quality mutant libraries is a powerful strategy for systematic analysis of gene essentiality and function. However, current genomic tools perform poorly for bacteriophages (phages) due to a lack of efficient selectable markers. Here we show a Tn5 -mediated e ssential g ene i nsertion m utagenesis (Tn5-EGIM) approach, which employs an essential phage gene as the selection marker for transposon construction and performs transposition within this essential gene-deficient phage genome, allowing plaque formation only if transposon insertions land in non-essential genomic regions. This strategy achieves 100% transposition efficiency and simplifies mutant library construction. Using Tn5-EGIM, we confirm known non-essential genes in phage T7 and identify twenty previously uncharacterized non-essential genes in phage T1. Cumulative gene deletion yields a streamlined T1 chassis with a 16% genome reduction and expands foreign DNA capacity. An engineered T1 phage built on T1 chassis infects T1-resistant Escherichia coli and exhibits enhanced antibacterial activity. Tn5-EGIM provides an efficient tool to dissect phage gene essentiality and advance engineered phage development.