PCR-based Screening of Targeted Mutants for the Fast and Simultaneous Identification of Bacterial Virulence Factors

Henriques, Ana Filipa Frutuoso Mendes; Carvalho, Frédéric A.; Pombinho, Rita; Reis, Olga; Sousa, Sandra; Cabanes, Didier

doi:10.2144/000113906

Cited by 10 publications

(5 citation statements)

References 16 publications

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“…Therefore, each mutant in a defined pool is labeled with a unique tag. For the present study, in order to simplify the detection of mutants present in both input and output pools, we developed a screening method based on PCR instead of the classical hybridization method previously used (19,20). This screening method is easier and less time-consuming than hybridization procedures.…”

Section: Resultsmentioning

confidence: 99%

Identification of Genes Involved in Neisseria meningitidis Colonization

et al. 2013

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Neisseria meningitidis is a worldwide cause of meningitis and septicemia leading at least to 50,000 deaths every year. Nevertheless, N. meningitidis is also a commensal bacterium that asymptomatically colonizes the epithelial cells of the nasopharynx of 10 to 30% of healthy individuals. Occasionally, N. meningitidis crosses the nasopharyngeal barrier and enters the bloodstream. During bacteremia, N. meningitidis may adhere to endothelial cells of brain vessels and invade meninges. To identify the genes required for meningococcal host colonization, we screened a signature-tagged transposon mutagenesis library using an innovative in vitro colonization model in order to identify mutants displaying decreased capacity to colonize human epithelial cells. Approximately 1,600 defined insertion mutants of invasive serogroup C strain NEM8013 were screened. Candidate mutants were tested individually for quantification of bacterial biomass with confocal microscope and COMSTAT software. Five mutants were demonstrated to exhibit significantly decreased colonization ability. The identified genes, including narP and estD, appeared to be involved in adaptation to hypoxic conditions and stress resistance. Interestingly, the genes fadD1, nnrS, and NMV_2034 (encoding a putative thioredoxin), prior to this study, had not been shown to be involved in colonization. Therefore, we provide here insights into the meningococcal functions necessary for the bacterium to adapt to growth on host cells.

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Section: Resultsmentioning

confidence: 99%

Identification of Genes Involved in Neisseria meningitidis Colonization

et al. 2013

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“…An uncleaved band would be shown if mutation occurred, and then mutation frequency was calculated by dividing uncleaved band intensity to the total band intensity from a single digestion experiment ( Fig. 2A) (Guschin et al, 2010;Henriques et al, 2012). Band intensity was measured using Quantity One Software (Bio-Rad, Hercules, CA, USA).…”

Section: Cas9-mrna/sgrna Microinjection and Mutation Analysismentioning

confidence: 99%

CRISPR/Cas9‐mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis

et al. 2017

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Pheromone-binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo. In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target-gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBP1-sgRNA injected eggs and 19.5% for PBP3-sgRNA injected eggs. Second, by an in-crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened, and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA-injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species.

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“…The positive clones were sequenced and then aligned with the wild-type sequences to determine whether they were mutated. In addition, the percentage of uncleaved band was measured by quantifying the band intensity with Quantity One Software (Bio-Rad) (Henriques et al 2012). The indel frequency was calculated by dividing uncleaved band intensity to the total band intensity from a single digestion experiment.…”

Section: Cas9 Messenger Rna In Vitro Transcriptionmentioning

confidence: 99%

Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

et al. 2014

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Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F 1 . Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species. R ECENTLY, a simple and efficient genome editing technology, type II CRISPR/Cas9, has been developed based on the Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas9) adaptive immune system. It requires three components for effective DNA cleavage: the nuclease Cas9, a targeting CRISPR RNA (crRNA), and an additional transactivating crRNA (tracrRNA) (Gasiunas et al. 2012;Jinek et al. 2012;Cho et al. 2013;Cong et al. 2013;Hwang et al. 2013;Mali et al. 2013). Further improvement of the system was achieved by fusing the crRNA and tracrRNA to form a single guide RNA (gRNA) that is sufficient to direct Cas9-mediated target cleavage (Hwang et al. 2013). Importantly, previous studies performed in vitro (Jinek et al. 2012), in bacteria , and in human cells (Cong et al. 2013) have shown that Cas9-mediated cleavage can be abolished by single mismatch at the gRNA-target site interface, particularly in the last 10-12 nucleotides located in the 39 end of the 20-nt gRNA targeting region. Compared to the other two engineered nuclease genome-editing technologies, zinc-finger nucleases (ZFNs) (Urnov et al. 2005;Doyon et al. 2008) and transcription activator-like effector nucleases (TALENs) (Huang et al. 2011;Sander et al. 2011;Tesson et al. 2011), the CRISPR/Cas9 system is substantially less expensive and much easier to program for editing new target sites. This new approach has been widely used for genome engineering in model animals, including Caenorhabditis elegans (Dickinson et al. 2013;Friedland et al. 2013;Tzur et al. 2013), Drosophila (Bassett et al. 2013;Ren et al. 2013;Yu et al. 2013), zebrafish (Chang et al. 2013Hrusc...

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PCR-based Screening of Targeted Mutants for the Fast and Simultaneous Identification of Bacterial Virulence Factors

Cited by 10 publications

References 16 publications

Identification of Genes Involved in Neisseria meningitidis Colonization

Identification of Genes Involved in Neisseria meningitidis Colonization

CRISPR/Cas9‐mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis

Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

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