Cell-free synthesis of infective phages from in vitro assembled phage genomes for efficient phage engineering and production of large phage libraries

Bacteriophages are promising alternatives to traditional antimicrobial treatment of bacterial infections. To further increase the potential of phages, efficient engineering methods are needed. This study investigates an approach to phage engineering based on phage rebooting and compares selected methods of assembly and rebooting of phage genomes. GG assembly of phage genomes and subsequent rebooting by cell-free transcription-translation reactions yielded the most efficient phage engineering and allowed production of a proof-of-concept T7 phage library of 1.8 × 10⁷ phages. We obtained 7.5 × 10⁶ different phages, demonstrating generation of large and diverse libraries suitable for high-throughput screening of mutant phenotypes. Implementing versatile and high-throughput phage engineering methods allows vastly accelerated and improved phage engineering, bringing us closer to applying effective phages to treat infections in the clinic.