REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

Trehan, Ashutosh; Kiełbus, Michał; Czapiński, Jakub; Stepulak, Andrzej; Huhtaniemi, Ilpo; Rivero-Müller, Adolfo

doi:10.1038/srep19121

Cited by 37 publications

(42 citation statements)

References 36 publications

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“…All mutants were generated by REPLACR technology and were sequence-verified (Trehan et al 2016). Primers for mutagenesis are presented in Supplementary Tables 1 and 2.…”

Section: Mutagenesismentioning

confidence: 99%

A vital region for human glycoprotein hormone trafficking revealed by an LHB mutation

Potorac

Rivero-Müller

Trehan

et al. 2016

Journal of Endocrinology

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Glycoprotein hormones are complex hormonally active macromolecules. Luteinizing hormone (LH) is essential for the postnatal development and maturation of the male gonad. Inactivating Luteinizing hormone beta (LHB) gene mutations are exceptionally rare and lead to hypogonadism that is particularly severe in males. We describe a family with selective LH deficiency and hypogonadism in two brothers. DNA sequencing of LHB was performed and the effects of genetic variants on hormone function and secretion were characterized by mutagenesis studies, confocal microscopy and functional assays. A 20-year-old male from a consanguineous family had pubertal delay, hypogonadism and undetectable LH. A homozygous c.118_120del (p.Lys40del) mutation was identified in the patient and his brother, who subsequently had the same phenotype. Treatment with hCG led to pubertal development, increased circulating testosterone and spermatogenesis. Experiments in HeLa cells revealed that the mutant LH is retained intracellularly and showed diffuse cytoplasmic distribution. The mutated LHB heterodimerizes with the common alpha-subunit and can activate its receptor. Deletion of flanking glutamic acid residues at positions 39 and 41 impair LH to a similar extent as deletion of Lys40. This region is functionally important across all heterodimeric glycoprotein hormones, because deletion of the corresponding residues in hCG, follicle-stimulating hormone and thyroid-stimulating hormone beta-subunits also led to intracellular hormone retention. This novel LHB mutation results in hypogonadism due to intracellular sequestration of the hormone and reveals a discrete region in the protein that is crucial for normal secretion of all human glycoprotein hormones.

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“…All mutants were generated by REPLACR technology and were sequence-verified (Trehan et al 2016). Primers for mutagenesis are presented in Supplementary Tables 1 and 2.…”

Section: Mutagenesismentioning

confidence: 99%

A vital region for human glycoprotein hormone trafficking revealed by an LHB mutation

Potorac

Rivero-Müller

Trehan

et al. 2016

Journal of Endocrinology

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“…The primers for insert amplification were KI-F and KI-R whereas the pair used for backbone linearization were BCB-F and BCB-R (Supplementary data, Table 1). Mutagenesis was performed by REPLACR methodology [19], using the SDM-F and SDM-R primers (Suppl material Table 1).…”

Section: Methodsmentioning

confidence: 99%

Genetical engineered lung cancer cell for analyzing Epithelial-Mesenchymal transition

Kiełbus

Czapiński

Kałafut

et al. 2019

Preprint

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Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible 12 manner, is a physiological processes that also exert important roles in disease progression Two forms 13 of cellular plasticity are epithelial-mesenchymal transition (EMT) and its inverse process, 14 mesenchymal-epithelial transition (MET). These processes have been correlated to the poor outcome 15 of different types of neoplasias as well as drug resistance development. Since EMT/MET are 16 transitional processes, we have generated and validated a reporter cell line. Specifically, a far-red 17 fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) 18 in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT 19 and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform 20 to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in the 21 future in vivo.22 Keywords: epithelial-mesenchymal transition (EMT); mesenchymal-epithelial transition (MET); 23 cancer cell line; vimentin; reporter; 24 25 29 transition (EMT) and mesenchymal-epithelial transition (MET), respectively. EMT has been 30 correlated to the metastasis and invasion potential of many types of malignancies [2]. The fact that 31 the poor outcome of many types of neoplasias correlates with EMT/MET, makes these molecular 32 phenomena important focus for research and drug targeting [3-6]. Despite the clinical association,33 the role of EMT/MET in metastasis is inconclusive, for example mesenchymal-like prostate cancer 34 cells survive in circulation but, unlike epithelial or cells undergoing EMT, they are unable to form 35 macrometastases [7]. In fact, breast cancer cell metastasis to lung tissue in mice was not affected by36 decreasing EMT -by targeting EMT-triggering transcription factors such as SNAI1 (Snail) and SNAI2 37 (Slug) by overexpressing miR-200 [8]. An additional role of EMT in tumorigenesis might be the 38 development of apoptotic tolerance and increased resistance to chemotherapy as has been found in 39animal models and patients [8,9]. Recently, a hybrid stage between epithelial and mesenchymal 40 2 of 19 phenotypes (hybE/M) has been recognized, and such hybE/M cells migrate outside the primary 41 tumors displaying some mesenchymal features such as spindle-like morphology, increased nuclear 42 levels of ZEB1 transcription factor, and epithelial ones such as cell-cell adhesion potential [10,11]. 43In order to study the transitory stages of EMT, MET and hybE/M, in particular at in vivo settings, 44 there is need to generate reporter cells to visualize phenotypical changes. There have been different 45 approaches to this, for example the use of heterologous fluorescent proteins driven under stage-46 specific promoters such as mesenchymal (ZEB1) and epithelial (CDH1) [12], mesenchymal snai1 and 47 epithelial sox10 in zebrafish [13], or mesenchymal Vimentin (Vim) in mice [14]. Nevertheless, 48 hete...

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“…Editing the ompA gene of E. coli and Enterobacter was complete as described in Reisch and Prather [35]. The protospacer sequence for the ompA gene was designed using the CHOPCHOP [74, 75], and cloned into pKDsgRNA-ack plasmid [35] directly upstream of gRNA scaffold using REPLACR mutagenesis protocol [76]. Two protospacer sequences were designed for each gene and the one which had lower escape rate after plating with or without aTC (S1 Table).…”

Section: Methodsmentioning

confidence: 99%

CRISPR/Cas9-mediated gene deletion of the ompA gene in symbiotic Enterobacter impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes

Hegde

Nilyanimit

Kozlova

et al. 2018

Preprint

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BackgroundSymbiotic bacteria are pervasive in mosquitoes and their presence can influence many host phenotypes that affect vectoral capacity. While it is evident that environmental and host genetic factors contribute in shaping the microbiome of mosquitoes, we have a poor understanding regarding how bacterial genetics affects colonization of the mosquito gut. The CRISPR/Cas9 gene editing system is a powerful tool to alter bacterial genomes facilitating investigations into host-microbe interactions but has yet to be applied to insect symbionts.Methodology/Principal FindingsTo investigate the role of bacterial factors in mosquito biology and in colonization of mosquitoes we used CRISPR/Cas9 gene editing system to mutate the outer membrane protein A (ompA) gene of an Enterobacter symbiont isolated from Aedes mosquitoes. The ompA mutant had an impaired ability to form biofilms and poorly infected Ae. aegypti when reared in a mono-association under gnotobiotic conditions. In adults the mutant had a significantly reduced infection prevalence compared to the wild type or complement strains, while no differences in prevalence were seen in larvae, suggesting bacterial genetic factors are particularly important for adult gut colonization. We also used the CRISPR/Cas9 system to integrate genes (antibiotic resistance and fluorescent markers) into these symbionts genome and demonstrated that these genes were functional in vitro and in vivo.Conclusions/SignificanceOur results shed insights onto the role of ompA gene in host-microbe interactions in Ae. aegypti and confirm that CRISPR/Cas9 gene editing can be employed for genetic manipulation of non-model gut microbes. The ability to use this technology for site-specific integration of genes into the symbiont will facilitate the development of paratransgenic control strategies to interfere with arboviral pathogens such Chikungunya, dengue, Zika and Yellow fever viruses transmitted by Aedes mosquitoes.Author summaryMicrobiota profoundly affect their host but few studies have investigated the role of bacterial genetics in host-microbe interactions in mosquitoes. Here we applied the CRISPR/Cas9 gene editing system to knock out a membrane protein in Enterobacter, which is a dominant member of the mosquito microbiome. The mutant strain lacked the capacity to form biofilms, infected larvae and adults at lower titers, and had a reduced prevalence in adults. The lower prevalence in adults, but not larvae, likely reflects the difference in the modes of bacterial acquisition from the larval water of these two life stages. Importantly from an applied perspective, we also demonstrated that this editing technology can be harnessed for site-specific integration of genes into the bacterial chromosome. In proof-of-principle studies we integrated either a fluorescent protein or gene conferring antibiotic resistance into the bacterial genome and showed these transgenes were functional in mosquitoes. The specificity, flexibility, and simplicity of this editing approach in non-model bacteria will be useful for developing novel symbiotic control strategies to control arthropod-borne disease.

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REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

Cited by 37 publications

References 36 publications

A vital region for human glycoprotein hormone trafficking revealed by an LHB mutation

A vital region for human glycoprotein hormone trafficking revealed by an LHB mutation

Genetical engineered lung cancer cell for analyzing Epithelial-Mesenchymal transition

CRISPR/Cas9-mediated gene deletion of the ompA gene in symbiotic Enterobacter impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes

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