An efficient, scarless, selection-free technology for phage engineering

Most recently developed phage engineering technologies are based on the CRISPR-Cas system. Here we present a non-CRISPR-based method for genetically engineering the Escherichia coli phages T5, T7, P1vir, and lambda vir by adapting the pORTMAGE technology, which was developed for engineering bacterial genomes. The technology comprises E. coli harboring a plasmid encoding a potent recombinase and a gene transiently silencing a repair system. Oligonucleotides with the desired phage mutation are electroporated into E. coli followed by infection of the target bacteriophage. The high efficiency of this technology, which yields 1-14% of desired recombinants, allows low-throughput screening for the desired mutant. We have demonstrated the use of this technology for single-base substitutions, for deletions of 50-201 bases, for insertions of 20 bases, and for 4 different phages. The technology may also be readily modified for use across many additional bacterial and phage strains.