Characterizing the portability of phage-encoded homologous recombination proteins

Abstract

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 ssDNA annealing proteins (SSAPs) improve HR 1000-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 if compatibility with this host domain is maintained. Furthermore, we find that co-expressing SSAPs with a paired SSB can significantly improve activity, 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 inaccessible through sequential nucleotide conversions.

Resolving the structure of phage–bacteria interactions in the context of natural diversity

“…Redβ/RecT-, ERF-, and Sak-families) are common in temperate phages 44 , and are thought to play an important role in their extensive genome modularity and mosaicism 45 . Such recombinases have been shown to be associated with large-scale recombination events of up to 79% genome length between incoming temperate phages and resident prophages 46 and are useful tools for in vitro genetic engineering (recombineering) [47][48][49][50] as they can facilitate recombination between sequence regions with as little as 23-bp sequence identity 51 . Noting a number of putative SSAP recombinase genes in our initial annotations, we sought to more systematically evaluate their representation in our diverse collection of phages.…”

ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida

“… 25 Besides recombinase expression levels, recent studies highlighted additional interactions with single-stranded DNA-binding proteins (SSBs) within the replication fork as a way to improve recombineering efficiency in a given host. 60 , 61 On the basis of results reported in other Pseudomonas species, coexpression of these SSBs together with recombinases might be an option for enhancing the allelic replacement efficiencies even further. 62 …”

DNA Damage Protection for Enhanced Bacterial Survival Under Simulated Low Earth Orbit Environmental Conditions in Escherichia coli

“…Indeed, it has been shown recently that in order to transfer effectively recombinogenic properties further modifications are required. For instance, compatible pairs of recombinases (phage origin in this case) and single stranded binding proteins (SSB) were required (Filsinger et al, 2021 ). RecA has a very close and dynamic relationship with SSB, displacement of the latter is modulated by RecA C-terminal (Eggler et al, 2003 ).…”