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

Moran G. Goren, Tridib Mahata, Udi Qimron*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

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 presented.).

Original languageEnglish
Pages (from-to)830-835
Number of pages6
JournalRNA Biology
Volume20
Issue number1
DOIs
StatePublished - 2023

Funding

FundersFunder number
European Research Council
Ministry of Health, State of Israel15370
Horizon 2020818878

    Keywords

    • Bacteriophages
    • DNA engineering
    • pORTMAGE

    Fingerprint

    Dive into the research topics of 'An efficient, scarless, selection-free technology for phage engineering'. Together they form a unique fingerprint.

    Cite this