Characterizing the portability of phage-encoded homologous recombination proteins

Gabriel T. Filsinger*, Timothy M. Wannier, Felix B. Pedersen, Isaac D. Lutz, Julie Zhang, Devon A. Stork, Anik Debnath, Kevin Gozzi, Helene Kuchwara, Verena Volf, Stan Wang, Xavier Rios, Christopher J. Gregg, Marc J. Lajoie, Seth L. Shipman, John Aach, Michael T. Laub, George M. Church*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review


Efficient genome editing methods are essential for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but techniques that rely on host RecA-mediated pathways are inefficient and laborious. Phage-encoded single-stranded DNA annealing proteins (SSAPs) improve HR 1,000-fold above endogenous levels. However, they are not broadly functional. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus and Caulobacter crescentus, we investigated the limited portability of SSAPs. We find that these proteins specifically recognize the C-terminal tail of the host’s single-stranded DNA-binding protein (SSB) and are portable between species only if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with SSBs can significantly improve genome editing efficiency, in some species enabling SSAP functionality even without host compatibility. Finally, we find that high-efficiency HR far surpasses the mutational capacity of commonly used random mutagenesis methods, generating exceptional phenotypes that are inaccessible through sequential nucleotide conversions. [Figure not available: see fulltext.]

Original languageEnglish
JournalNature Chemical Biology
Issue number4
Pages (from-to)394-402
Publication statusPublished - Apr 2021

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