Programming virulent bacteriophages by developing a multiplex genome engineering method

Unlike temperate phages, which integrate into host genomes and allow time for genetic manipulation, lytic phages rapidly hijack the bacterial machinery and trigger host lysis within minutes, leaving an extremely narrow editing window. Furthermore, virulent phage genes usually encode toxic products that inhibit the growth of bacterial hosts. We developed a SMART (splitting, modifying, assembling, and rebooting) method for multiplex genome engineering of virulent phages. We deleted 3.9 kb sequences distributed across 8 sites in the 39.9 kb genome of the wild-type T7 <i>Escherichia coli</i> phage to construct a chassis phage. Synthetic T7 phages expressing heterologous lysin genes were constructed to efficiently lyse different bacteria, such as <i>E. coli</i>, <i>Staphylococcus aureus,</i> or <i>Streptococcus agalactiae</i>. The SMART method will facilitate targeted modifications of phage genomes for the creation of custom-designed phages with enhanced therapeutic efficacy, broader host specificity, and programmable behaviors.