An efficient CRISPR-Cas12a tool for iterative genome editing, streamlined minimization, and payload engineering of...

The ability to precisely engineer phage genomes is critical for advancing phage-based biotechnology and therapeutic development. Here, we present a high-efficiency, scarless CRISPR-Cas12a genome editing method for a virulent Pseudomonas aeruginosa phage vB_PaeM_SCUT-S1 (S1). Using a two-plasmid system, we achieved near-complete efficiency in gene deletion, point mutation, gene insertion, and replacement. Iterative deletions enabled the identification of 27 non-essential, 16 quasi-essential, and seven essential genes, and culminated in the largest genome reduction (13.9 kb) reported for a P. aeruginosa phage. The minimized phage mutant S1_200L retained infectivity and supported integration of large exogenous gene cassettes (e.g., lacZ, lys009). This system offers a versatile and high-throughput approach for phage genome engineering and rational design of functional phages for synthetic biology and antimicrobial applications.