Defining the essential genome of diverse phages with phage Tn-seq

Key Points:

• Researchers developed a cloning strategy using Golden Gate assembly to insert spacers targeting phage JS26 into a selection plasmid, enabling efficient targeting and mutagenesis studies.
• Transposon mutagenesis combined with Cas13a counter-selection allowed identification of essential genes in phages JS26 and PCH45 by enriching for transposon-containing mutants resistant to CRISPR interference.
• Analysis of transposon insertion sites and RNA-seq data revealed gene essentiality patterns, with essential genes showing fewer insertions and correlations with expression levels during infection.
• Fusion of the transposase with UvsX protein enhanced mutagenesis efficiency in PCH45, and microscopy confirmed localization of UvsX to phage nucleus DNA, supporting its role in transposon insertion.
• Comprehensive datasets including transposon insertion sequencing, proteomics, and RNA-seq provide detailed gene essentiality profiles and functional annotations for phages JS26 and PCH45, contributing to understanding of phage biology and genome organization.