Abstract
Phages provide a potential therapy for multi-drug-resistant (MDR) bacteria. However, a significant portion of viral genes often remains unknown, posing potential dangers. The identification of non-essential genes helps dissect and simplify phage genomes, but current methods have various limitations. In this study, we present an in vivo two-plasmid transposon insertion system to assess the importance of phage genes, which is based on the V. cholerae transposon Tn6677, encoding a nuclease-deficient type I-F CRISPR-Cas system. We first validated the system in Pseudomonas aeruginosa PAO1 and its phage S1. We then used the selection marker AcrVA1 to protect transposon-inserted phages from CRISPR-Cas12a and enriched the transposon-inserted phages. For a pool of selected 10 open-reading frames (2 known functional protein genes and 8 hypothetical protein genes) of phage S1, we identified 5 (2 known functional protein genes and 3 hypothetical protein genes) as indispensable genes and the remaining 5 (all hypothetical protein genes) as dispensable genes. This approach offers a convenient, site-specific method that does not depend on homologous arms and double-strand breaks (DSBs), holding promise for future applications across a broader range of phages and facilitating the identification of the importance of phage genes and the insertion of genetic cargos.