Improved bacterial recombineering by parallelized protein discovery

Abstract

<jats:p>Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found. Here we introduce a high-throughput method for SSAP discovery that we call “serial enrichment for efficient recombineering” (SEER). By performing SEER in<jats:italic>Escherichia coli</jats:italic>to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5- to 10-fold in<jats:italic>E. coli</jats:italic>, while PapRecT enables efficient recombineering in<jats:italic>Pseudomonas aeruginosa</jats:italic>, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.</jats:p>

Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria

“…The main focus to boost recombination frequencies of MAGE has been put on the recombinase itself, as the state-of-the-art Red-β protein for recombineering in Enterobacteria shows varying functionality in other hosts 153 . Superior ssDNA recombinases have been identified from prophages for P. putida 89 , K. pneumoniae 89 , B. subtilis 154 , L. lactis 22 , and M. smegmatis 153 , but the recombination frequencies remain well below those reported for E. coli , which in turn limits large-scale, multiplexed engineering efforts. Moreover, the use of alternative recombinases requires time-consuming optimization of recombinase expression levels and oligonucleotide design 21 , 89 , 153 , 154 .…”

“…To date, the use of these versatile enzymes in non-model hosts has mostly remained limited to genomic integration tools and recombineering. The characterization of existing and novel integrases and recombinases from prophages will optimize the currently available recombinase-based tools for these bacteria 89 . Additionally, they will enable the implementation of more advanced applications, including the construction of complex logic gates and memory devices, thus allowing non-model hosts to reach their full potential as valuable biological chassis for SynBio applications.…”

“…On a critical note, one potential limitation of phage-based parts and tools is that they are mostly species-specific, thus limiting portability to other species. However, recent examples indicate that some phage recombinases may display broad host specificity 89 . Rather than solving the portability issue within SynBio, we call for caution when using E. coli -optimized tools blindly in other hosts and promote the development of host-specific toolboxes instead.…”

Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects

“…While it is potentially difficult to engineer microbial SSAPs to function in mammalian contexts, they have been shown to harbor significant metagenomic diversity with distinctive activities depending on host organisms ( 49 , 50 , 53 , 66 , 67 ). Hence, we began our search for gene-editing phage proteins by examining different families of phage SSAPs ( 50 , 68 ).…”

“…The simplicity and efficiency of phage SSAP provide intriguing possibilities for gene-editing in higher eukaryotes. To date, many studies have leveraged SSAPs for in vitro and bacterial applications, they have not been used for mammalian gene editing ( 46 , 49 ). We hypothesized that SSAP may facilitate homology-directed genome editing in mammalian cells when coupled to programmable Cas9 for genomic targeting (Figure 1A ).…”