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, P1, and λ by adapting the pORTMAGE technology, which was developed for engineering bacterial genomes. The technology comprises E. coli harbouring 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 four different phages. The technology may also be readily modified for use across many additional bacterial and phage strains.[Figure: see text].