Cell-Free Engineering of Synthetic Phages to Overcome Pseudomonas Aeruginosa Resistance
Description
Multidrug-resistant Pseudomonas aeruginosa is a major global health threat, demanding innovative therapeutic solutions beyond traditional antibiotics. Bacteriophages, viruses that infect bacteria, offer a promising alternative. Traditional phage engineering approaches rely on bacterial hosts for genome replication, mutation screening, and functional validation, leading to longer experimental timelines, unpredictable recombination events, and host dependent constraints. To overcome these limitations, we present a novel approach to rationally design and construct synthetic phages with reduced genomes to evade bacterial defense mechanisms. Starting from a de novo synthesized wild-type phage genome, we systematically eliminate nonessential regions while integrating targeted mutations to bypass bacterial resistance, including CRISPR-Cas and restriction-modification barriers. Unlike conventional approaches, our method employs a cell-free transcription translation (TXTL) system for rapid genome prototyping, precise modifications, and predictable engineering. This approach eliminates the host constraints by in vitro phage genome assembly and functional validation, facilitating iterative design and precise genetic modifications before host introduction. Our method improves precision, efficiency, and reproducibility in phage engineering by overcoming bacterial propagation constraints and establishing a next generation framework for targeted phage therapies.