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].

Reporting off-target effects of recombinant engineering using the pORTMAGE system.

pORTMAGE recombineering is a simple technique for incorporation of novel point mutations into bacterial genomes that eliminates off-target effects. Here we inserted point mutations into the cusS gene from Escherichia coli, then, using Illumina sequencing, report genetic variants in all mutant strains. Several off-site mutations were found at high frequency. Low frequency mutations also show high heterogeneity. This means that it is essential for studies to report all off-target effects and acknowledge the effect that this may have on resultant phenotypes.