Harnessing stepping-stone hosts to engineer, select, and reboot synthetic bacteriophages in one pot

Advances in synthetic genomics have led to a great demand for genetic manipulation. Trimming any process to simplify and accelerate streamlining of genetic code into life holds great promise for synthesizing and studying organisms. Here, we develop a simple but powerful stepping-stone strategy to promote genome refactoring of viruses in one pot, validated by successful cross-genus and cross-order rebooting of 90 phages infecting 4 orders of popular pathogens. Genomic sequencing suggests that rebooting outcome is associated with gene number and DNA polymerase availability within phage genomes. We integrate recombineering, screening, and rebooting processes in one pot and demonstrate genome assembly and genome editing of phages by stepping-stone hosts in an efficient and economic manner. Under this framework, in vitro assembly, yeast-based assembly, or genetic manipulation of native hosts are not required. As additional stepping-stone hosts are being developed, this framework will open doors for synthetic phages targeting more pathogens and commensals.