Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases

Mosberg, Joshua A.; Gregg, Christopher; Lajoie, Marc J.; Wang, Harris He; Church, George M.

doi:10.1371/journal.pone.0044638

Cited by 83 publications

(85 citation statements)

References 51 publications

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“…We expect that optimized protocols involving several cycles would produce results closer to those seen with Recβ in E. coli . Further, the P. putida EM42 cell factory, while purged of certain endonucleases (Martínez‐García et al ., 2014) that could inhibit strand displacement (Mosberg et al ., 2012), harbours a fully functional MMR system. As mentioned, MMR machinery is known to interfere with the MAGE platform in various bacterial species, dampening final rates of mutagenesis.…”

Section: Discussionmentioning

confidence: 99%

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

Ricaurte

Martínez‐García

Nyerges

et al. 2017

Microbial Biotechnology

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SummaryBacterial recombineering typically relies on genomic incorporation of synthetic oligonucleotides as mediated by Escherichia coli λ phage recombinase β – an occurrence largely limited to enterobacterial strains. While a handful of similar recombinases have been documented, recombineering efficiencies usually fall short of expectations for practical use. In this work, we aimed to find an efficient Recβ homologue demonstrating activity in model soil bacterium Pseudomonas putida EM42. To this end, a genus‐wide protein survey was conducted to identify putative recombinase candidates for study. Selected novel proteins were assayed in a standardized test to reveal their ability to introduce the K43T substitution into the rpsL gene of P. putida. An ERF superfamily protein, here termed Rec2, exhibited activity eightfold greater than that of the previous leading recombinase. To bolster these results, we demonstrated Rec2 ability to enter a range of mutations into the pyrF gene of P. putida at similar frequencies. Our results not only confirm the utility of Rec2 as a Recβ functional analogue within the P. putida model system, but also set a complete workflow for deploying recombineering in other bacterial strains/species. Implications range from genome editing of P. putida for metabolic engineering to extended applications within other Pseudomonads – and beyond.

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Section: Discussionmentioning

confidence: 99%

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

Ricaurte

Martínez‐García

Nyerges

et al. 2017

Microbial Biotechnology

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“…They found that the use of an E. coli strain deleted of four ssDNA exonucleases (RecJ, ExoI, ExoVII, and ExoX) resulted in higher amounts of mean allele conversions using the Co-MAGE protocol (374). The implication is that more oligos were surviving the threat of degradation by these exonucleases prior to being annealed to the replication fork.…”

Section: Mechanisms Of λ Red Recombineering Recombineering With Ssdnamentioning

confidence: 99%

λ Recombination and Recombineering

Murphy

2016

EcoSal Plus

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The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.

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“…E. coli degrades linear double-strand DNA very rapidly via the RecBCD and SbcCD exonucleases. Gam, one of the three proteins of , prevents the degradation of the linear DNA by inhibiting the nucleases, Exo generates single-stranded DNA, and Beta binds to this single-stranded DNA and facilitates recombination to homologous chromosomal regions (16). In Bacillus subtilis, such a system is not necessary.…”

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confidence: 99%

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Altenbuchner

2016

Appl Environ Microbiol

271

246

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The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system from Streptococcus pyogenes has recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing of Bacillus subtilis. The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication for Escherichia coli, the temperature-sensitive replication origin of plasmid pE194 ts for B. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries the cas9 gene under the control of the B. subtilis mannose-inducible promoter P manP and a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5= end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in the B. subtilis chromosome and by repair of the trpC2 mutation of B. subtilis 168. IMPORTANCEIn prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing of B. subtilis overcomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

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Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases

Cited by 83 publications

References 51 publications

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

λ Recombination and Recombineering

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

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