A new recombineering system for Photorhabdus and Xenorhabdus

Yin, Jia; Zhu, Hongbo; Xia, Liqiu; Ding, Xuezhi; Hoffmann, Thomas; Hoffmann, Michael; Bian, Xiaoying; Müller, Rolf; Fu, Jun; Stewart, A. Francis; Zhang, Youming

doi:10.1093/nar/gku1336

Cited by 59 publications

(67 citation statements)

References 52 publications

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“…Automated, high-throughput, and multiplexed genome editing applications have also been envisioned, including multiplexed automated genome engineering (MAGE), generating up to 4.3 billion genomic mutants per day (13). Finally, phage-mediated recombineering systems have been identified and exploited for use in an array of bacterial genera (14)(15)(16)(17)(18)(19).…”

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confidence: 99%

Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

Pyne

Moo‐Young

Chung

et al. 2015

Appl Environ Microbiol

211

188

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cTo date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and "scar"-less. Since its inception in 1998, phage recombinase-mediated homologous recombination, also known as recombineering, has revolutionized bacterial genetics, synthetic biology, and metabolic engineering (1, 2). Relying on either RecET from the Rac prophage (2) or the bacteriophage lambda Red proteins, Exo, Beta, and Gam (1), recombineering allows simple and efficient construction of gene knockout mutants via homologous recombination of a double-stranded DNA (dsDNA) PCR product with bacterial chromosomes (3). This work has led to the construction of an Escherichia coli K-12 single-gene knockout library (the Keio collection [4]), enabled widespread use of chromosome-encoded expression platforms for metabolic and strain engineering (5-11), and simplified conventional recombinant DNA technology through the advent of ligation-independent DNA cloning (2, 12). Automated, high-throughput, and multiplexed genome editing applications have also been envisioned, including multiplexed automated genome engineering (MAGE), generating up to 4.3 billion genomic mutants per day (13). Finally, phage-mediated recombineering systems have been identified and exploited for use in an array of bacterial genera (14-19).Recombineering can be carried out using either singlestranded DNA (ssDNA) or dsDNA substrates. Since recombineering occurs through an entirely ssDNA intermediate (20), ssDNA recombineering requires only an ssDNA recombinase (RecT or Beta) for recombination (21), whereas recombination of dsDNA substrates requires both an exonuclease (RecE or Exo) and its associated recombinase (RecT or Beta). For this reason, recombination of ssDNA substrates, such as oligonucleotides, is simpler, more efficient, and better understood ...

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mentioning

confidence: 99%

Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

Pyne

Moo‐Young

Chung

et al. 2015

Appl Environ Microbiol

211

188

View full text Add to dashboard Cite

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“…Recently, another method to perform chromosomal insertions into the P. luminescens genome has been developed based on a P. luminescens endogenous recombinase system that is homologous to lambda-red (15), which is a useful tool for integrating promoter insertions or gene knockouts into the genome using linear DNA fragments. However, since P. luminescens does not grow at 37C, it is challenging to remove the temperature-sensitive plasmid that carries the recombinase genes after the recombination event.…”

Section: Resultsmentioning

confidence: 99%

A Novel Tool for Stable Genomic Reporter Gene Integration to Analyze Heterogeneity in Photorhabdus Luminescens at the Single-Cell Level

Glaeser

Heermann

2015

BioTechniques

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Determination of reporter gene activity at the single-cell level is a prerequisite for analyzing heterogeneous gene expression in bacteria. The insect pathogenic enteric bacterium Photorhabdus luminescens is an excellent organism in which to study heterogeneity since it exists in two phenotypically different forms, called the primary and secondary variant. A tool for generating stable genomic integrations of reporter genes has been lacking for these bacteria, and this has hampered the acquisition of reliable data sets for promoter activities at the single-cell level. We therefore generated a plasmid tool named pPINT-mCherry for the easy and stable introduction of gene fragments upstream of an mCherry reporter gene followed by stable integration of the plasmid into the P. luminescens genome at the rpmE/glmS intergenic region. We demonstrate that the genomic integration of reporter genes for single-cell analysis is necessary in P. luminescens since plasmid-borne reporter genes mimic heterogeneity and are therefore not applicable in these bacteria, in contrast to their use in single-cell analysis in other bacteria like Escherichia coli.

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“…Recently, the generation of a recombineering system in the related genera, Photorhabdus and Xenorhabdus was reported [40]. Each harbors genes analogous to Red α, β, and γ, which have been repurposed for gene cluster activation by promoter exchange.…”

Section: Methodologiesmentioning

confidence: 99%

“…Each harbors genes analogous to Red α, β, and γ, which have been repurposed for gene cluster activation by promoter exchange. The study introduced a tetracycline inducible promoter, using the analogous λ Red system, upstream of a cryptic chromosomal NRPS gene cluster to activate expression, resulting in the production of nonribosomal peptides ranging from a heptapeptide to a nonapeptide in the native bacterium [40]. Although this study did not involve direct capture of gene clusters from genomic DNA, it expanded on the phage homologous recombination system with potentially broad implications.…”

Section: Methodologiesmentioning

confidence: 99%

Direct Capture Technologies for Genomics-Guided Discovery of Natural Products

Chan¹,

Maria²,

2016

CTMC

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Microbes are important producers of natural products, which have played key roles in understanding biology and treating disease. However, the full potential of microbes to produce natural products has yet to be realized; the overwhelming majority of natural product gene clusters encoded in microbial genomes remain “cryptic”, and have not been expressed or characterized. In contrast to the fast-growing number of genomic sequences and bioinformatic tools, methods to connect these genes to natural product molecules are still limited, creating a bottleneck in genome-mining efforts to discover novel natural products. Here we review developing technologies that leverage the power of homologous recombination to directly capture natural product gene clusters and express them in model hosts for isolation and structural characterization. Although direct capture is still in its early stages of development, it has been successfully utilized in several different classes of natural products. These early successes will be reviewed, and the methods will be compared and contrasted with existing traditional technologies. Lastly, we will discuss the opportunities for the development of direct capture in other organisms, and possibilities to integrate direct capture with emerging genome-editing techniques to accelerate future study of natural products.

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A new recombineering system for Photorhabdus and Xenorhabdus

Cited by 59 publications

References 52 publications

Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

A Novel Tool for Stable Genomic Reporter Gene Integration to Analyze Heterogeneity in Photorhabdus Luminescens at the Single-Cell Level

Direct Capture Technologies for Genomics-Guided Discovery of Natural Products

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