Lox’d in translation: contradictions in the nomenclature surrounding common lox-site mutants and their implications in experiments

Shaw, Daniel; Serrano, Luís; Lluch‐Senar, María

doi:10.1099/mic.0.000997

Cited by 3 publications

(3 citation statements)

References 63 publications

(89 reference statements)

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“…The first template was made of (i) a centromere (CEN6), an autonomously replicating sequence (ARSH4), and the HIS3 selection marker for selection in yeast, (ii) a gentamicin resistance marker for selection in mycoplasma cells, and (iii) 60 bp recombination arms placed at each extremity, identical to the sequences flanking the target gene. The second template was indistinguishable from the first except for the presence of two 34 bp loxP sites [ 55 ] flanking the Cre targets, i.e. the gentamicin resistance marker and the yeast components that must be removed to obtain seamless mutants (Fig.…”

Section: Resultsmentioning

confidence: 99%

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A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae

Gourgues,

Manso-Silván,

Chamberland

et al. 2024

Microbiology

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Mycoplasma capricolum subspecies capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for Mccp genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to Mccp. CReasPy-Cloning was used to simultaneously clone and engineer the Mccp genome in yeast, prior to whole-genome transplantation into M. capricolum subsp. capricolum recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific Mccp mutants. The Cre–lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in Mccp allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of Mccp and the rational design of novel, improved vaccines for the control of CCPP.

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Section: Resultsmentioning

confidence: 99%

“…S1, available in the online version of this article) using the primer pairs MccpPeptS41pMT85gentPRS-F/MccpPeptS41pMT85gentPRS-F. The second one was produced with the primer pairs MccpPeptS41LOX66pMT85gentPRS-F/MccpPeptS41LOX71pMT85gentPRS-R in order to include loxP sites [ 55 ] in the amplicon (Fig. S2).…”

Section: Methodsmentioning

confidence: 99%

A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae

Gourgues,

Manso-Silván,

Chamberland

et al. 2024

Microbiology

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show abstract

“…The RBEs are separated by an 8-bp asymmetric spacer that determines the orientation of each site, and the outcome of a recombination reaction. [8][9][10][11] Four molecules of Cre assemble with two loxP DNA sites and, in alternating fashion, tyrosine residues of each protomer perform a series of strand cleavages, exchanges and ligations, forming covalent 3'-phosphotyrosine and Holliday junction intermediates in order to generate recombinant products. Cre catalyzes this reaction without the need of additional factors or consumption of ATP, and thus, has emerged as a powerful tool for genome engineering in the laboratory.…”

Section: Introductionmentioning

confidence: 99%

Unloxing the assembly and activation mechanism of Cre recombinase using Cryo-EM

Stachowski

Norris

Potter

et al. 2021

Preprint

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Mechanistic understanding of the structural basis for DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes (intasomes). These structural and biochemical studies have suggested that conformational changes and DNA bending in presynaptic complexes underlie site-selection and activation mechanisms of Cre recombinase. Here we used protein engineering and various DNA substrates to isolate the Cre-loxP (54 kDa), Cre2-loxP (110 kDa), and Cre4-loxP2 assembly intermediates, and determined their structures using cryo-EM to resolutions of 3.9 Å, 4.5 Å, and 3.2 Å, respectively. Progressive DNA bending along the assembly pathway enables formation of increasingly intimate protein-protein interfaces. Insufficient stabilization of important protein motifs observed during the assembly process provides a compelling explanation for the observed half-the-sites activity, and preferential bottom strand cleavage of loxP sequences. We found that selection of loxP sites is largely dependent on the ability for Cre to bend and stabilize the spacer region between two recombinase binding elements. Application of 3D variability analysis to the tetramer data reveals a propensity for motion along the pathway between protomer activation and Holliday junction isomerization. These findings help us to better understand loxP site specificity, controlled activation of alternating protomers, the basis for the observed bias of strand cleavage order, and the importance of conformational sampling, especially with regards to site-selection and activity among Cre variants. Furthermore, our findings provide invaluable information for the rational development of designer, site-specific recombinases for use as gene editing technologies.

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Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM

Stachowski

Norris

Potter

et al. 2022

Nucleic Acids Research

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Cre recombinase selectively recognizes DNA and prevents non-specific DNA cleavage through an orchestrated series of assembly intermediates. Cre recombines two loxP DNA sequences featuring a pair of palindromic recombinase binding elements and an asymmetric spacer region, by assembly of a tetrameric synaptic complex, cleavage of an opposing pair of strands, and formation of a Holliday junction intermediate. We used Cre and loxP variants to isolate the monomeric Cre-loxP (54 kDa), dimeric Cre2-loxP (110 kDa), and tetrameric Cre4-loxP2 assembly intermediates, and determined their structures using cryo-EM to resolutions of 3.9, 4.5 and 3.2 Å, respectively. Progressive and asymmetric bending of the spacer region along the assembly pathway enables formation of increasingly intimate interfaces between Cre protomers and illuminates the structural bases of biased loxP strand cleavage order and half-the-sites activity. Application of 3D variability analysis to the tetramer data reveals constrained conformational sampling along the pathway between protomer activation and Holliday junction isomerization. These findings underscore the importance of protein and DNA flexibility in Cre-mediated site selection, controlled activation of alternating protomers, the basis for biased strand cleavage order, and recombination efficiency. Such considerations may advance development of site-specific recombinases for use in gene editing applications.

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Lox’d in translation: contradictions in the nomenclature surrounding common lox-site mutants and their implications in experiments

Cited by 3 publications

References 63 publications

A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae

A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae

Unloxing the assembly and activation mechanism of Cre recombinase using Cryo-EM

Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM

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