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 inEscherichia colito 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 inE. coli, while PapRecT enables efficient recombineering inPseudomonas aeruginosa, 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.

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.

14Exploiting bacteriophage-derived homologous recombination processes has enabled precise, 15 multiplex editing of microbial genomes and the construction of billions of customized genetic 16 variants in a single day. The techniques that enable this, Multiplex Automated Genome 17 Engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are 18 however currently limited to a handful of microorganisms for which single-stranded DNA-19 annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to 20 enable genome-scale engineering in new hosts, highly efficient SSAPs must first be found. Here 21 we introduce a high-throughput method for SSAP discovery that we call Serial Enrichment for 22 Efficient Recombineering (SEER). By performing SEER in E. coli to screen hundreds of putative 23 SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency 24 of single-locus editing to as high as 50% and improves multiplex editing by 5 to 10-fold in E. coli, 25while PapRecT enables efficient recombineering in Pseudomonas aeruginosa, a concerning 26 human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically 27 relevant enterobacteria. We envision that the deployment of SEER in new species will pave the 28 way toward pooled interrogation of genotype-to-phenotype relationships in previously 29 intractable bacteria. 30 31 bacteria that can contain billions of precisely targeted mutations. However, while these 48 techniques function well in E. coli and some closely related Enterobacteria, efforts to reproduce 49 these results in other bacterial species have been sporadic and stymied by low efficiencies (Table 50 S1) [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] . 51The incorporation of genomic modifications via oligonucleotide annealing at the replication fork, 52 called oligo-mediated recombineering, is the molecular mechanism that drives MAGE and 53 DIvERGE 4,32,33 . This method was first described in E. coli, and is most commonly promoted by the 54 expression of bet (here referred to as Redβ) from the Red operon of Escherichia phage λ 5,6,34 . 55 Redβ is a single-stranded DNA-annealing protein (SSAP) whose role in recombineering is to 56 anneal ssDNA to complimentary genomic DNA at the replication fork. Though improvements to 57 recombineering efficiency have been made 7-9 , the core protein machinery has remained 58 constant, with Redβ representing the state-of-the-art in E. coli and enterobacterial 59 recombineering. Redβ additionally does not adapt well to use outside of E. coli, displaying host 60 tropism, presumably toward hosts that are targets of infection for Escherichia phage λ. To enable 61 recombineering in organisms in which Redβ does not work efficiently, most often Redβ homologs 62 from prophages of genetically similar bacteria are screened 15,16,21,23,30 , but high levels of 63 recombineering efficiency, as seen in E. coli, remain elusive. 64We hypothesized tha...

Bacterial genome editing methods are used to engineer strains for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but traditional techniques using endogenous RecA-mediated pathways are inefficient and 5 laborious. Phage encoded RecT proteins can improve HR over 1000-fold, but these proteins have limited portability between species. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus, and Caulobacter crescentus we investigated the hostlimited functionality of RecTs. We find that these proteins specifically recognize the 7 Cterminal amino acids of the bacterial single-stranded DNA-binding protein (SSB), and are 10 portable between species only if compatibility with this host domain is maintained. Furthermore, in some species, we find that co-expressing otherwise incompatible RecTs with a paired bacterial SSB is sufficient to establish functionality. Finally, we demonstrate that high-efficiency HR surpasses the mutational capacity of more widely used error-prone methods for genome diversification, and can be used to identify exceptional phenotypes inaccessible through 15 sequential nucleotide conversions.