A5063341262- CRISPR and Genetic Engineering
- Bacterial Genetics and Biotechnology
- RNA and protein synthesis mechanisms
- Advanced biosensing and bioanalysis techniques
- DNA and Nucleic Acid Chemistry
- Antibiotic Resistance in Bacteria
- Bacteriophages and microbial interactions
- Bacterial biofilms and quorum sensing
- Animal Genetics and Reproduction
- Bacterial Identification and Susceptibility Testing
- Vibrio bacteria research studies
- RNA Interference and Gene Delivery
- Antibiotics Pharmacokinetics and Efficacy
- Genomics and Chromatin Dynamics
- Biochemical and Structural Characterization
- Silk-based biomaterials and applications
- Cytokine Signaling Pathways and Interactions
- Microbial Metabolic Engineering and Bioproduction
- Sirtuins and Resveratrol in Medicine
- Bone health and treatments
- Induction Heating and Inverter Technology
- Monoclonal and Polyclonal Antibodies Research
- Innovative Microfluidic and Catalytic Techniques Innovation
- Viral Infectious Diseases and Gene Expression in Insects
- Nanofabrication and Lithography Techniques
Changzhou University
2024
Institut Pasteur
2016-2023
Université Paris Cité
2022-2023
Centre National de la Recherche Scientifique
2022-2023
Jilin Electric Power Research Institute (China)
2021
The University of Adelaide
2013-2014
High-throughput CRISPR-Cas9 screens have recently emerged as powerful tools to decipher gene functions and genetic interactions. Here we use a genome-wide library of guide RNAs direct the catalytically dead Cas9 (dCas9) block transcription in Escherichia coli. Using machine-learning approach, reveal that sharing specific 5-nucleotide seed sequences can produce strong fitness defects or even kill E. coli regardless other 15 nucleotides sequence. This effect occurs at high dCas9 concentrations...
The RNA-guided Cas9 nuclease from CRISPR-Cas systems has emerged as a powerful biotechnological tool. specificity of can be reprogrammed to cleave desired sequences in cell's chromosome simply by changing the sequence small guide RNA. Unlike most eukaryotes, cleavage bacteria been reported kill cell. However, mechanism cell death remains investigated. Bacteria mainly rely on homologous recombination (HR) with sister chromosomes repair double strand breaks. Here, we show that simultaneous all...
We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases time and effort needed integration single or multiple fragments. Additionally, facilitates expression genetic elements are impossible propagate typical multicopy plasmids.
High-throughput genetic screens are powerful methods to identify genes linked a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used silence interest in method also known as CRISPRi. Here, we report genome-wide CRISPR-dCas9 screen using starting pool ~ 92,000 sgRNAs which target random positions chromosome E. coli. To benchmark our method, first investigate its utility predict gene essentiality genome coli during growth rich medium. We...
Abstract Genetic tools derived from the Cas9 RNA-guided nuclease are providing essential capabilities to study and engineer bacteria. While importance of off-target effects was noted early in Cas9’s application mammalian cells, cleavage by bacterial genomes is easily avoided due their smaller size. Despite this, several studies have reported experimental setups which expression toxic, even when using catalytic dead variant (dCas9). Specifically, dCas9 shown be toxic complex with guide RNAs...
Article8 March 2018Open Access Source DataTransparent process Tuning dCas9's ability to block transcription enables robust, noiseless knockdown of bacterial genes Antoine Vigouroux Synthetic Biology Laboratory, Institut Pasteur, Paris, France Microbial Morphogenesis and Growth Search for more papers by this author Enno Oldewurtel orcid.org/0000-0002-2813-0259 Lun Cui orcid.org/0000-0001-5907-0538 David Bikard Corresponding Author [email protected] orcid.org/0000-0002-5729-1211 Sven van...
Abstract Type II CRISPR-Cas systems introduce double-strand breaks into DNA of invading genetic material and use fragments to acquire novel spacers during adaptation. These can be the substrate several repair pathways, paving way for interactions. We report that non-homologous end-joining (NHEJ) type II-A only co-occur once among 5563 fully sequenced prokaryotic genomes. investigated experimentally possible molecular interactions using NHEJ pathway from Bacillus subtilis Streptococcus...
The ability to block gene expression in bacteria with the catalytically inactive mutant of Cas9, known as dCas9, is quickly becoming a standard methodology probe function, perform high-throughput screens, and engineer cells for desired purposes. Yet, we still lack good understanding design rules that determine on-target activity dCas9. Taking advantage screening data, fit model predict dCas9 RNA polymerase based on target sequence, validate its performance independently generated datasets....
Significance Proteins bound to DNA often interact with proteins elsewhere on the same regulate gene expression. The intervening tethers near each other, making their interaction efficient and specific, but importance of this tethering effect is poorly understood at large separations. We quantitated inside bacterial cells, using two different separations up 10,000 bp, show that strong enough drive interactions over these distances. were ∼10-fold weaker outside implying cellular factors...
Spider silk proteins (spidroins) have garnered attention in biomaterials research due to their ability self-assemble into hydrogels. However, reported spidroin hydrogels require high protein concentration and prolonged gelation time. Our study engineered an artificial that exhibits unprecedented rapid self-assembly at physiologically relevant conditions, achieving a low of 6 mg/mL 37 °C without external additives. Remarkably, 30 concentration, our forms within s, feature we termed “superfast...
How distant enhancer elements regulate the assembly of a transcription complex at promoter remains poorly understood. Here, we use long-range gene regulation by bacteriophage λ CI protein as powerful system to examine this process in vivo. A 2.3-kb DNA loop, formed bridging its binding sites OR and OL , is known already enhance repression lysogenic PRM located . show that looping also activates allowing C-terminal domain α subunit RNA polymerase bound contact site adjacent distal Our results...
In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of envelope permeability barrier to some antibiotics and detergents. Enterobacteria, stress caused by unfolded OMPs activates sigmaE (σE) transcriptional response. σE upregulates OMP biogenesis factors, including β-barrel assembly machinery (BAM) that catalyses folding. Here we report DolP (formerly YraP), a σE-upregulated poorly understood lipoprotein, is fitness in cells undergo stress. We...
Abstract High-throughput genetic screens are powerful methods to identify genes linked a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used silence interest in method also known as CRISPRi. Here, we report genome-wide CRISPR-dCas9 screen using pool ~ 92,000 sgRNAs which target random positions chromosome E. coli . We first investigate utility this for prediction essential and various unusual features genome then apply discover required...
Abstract The main outcome of efficient CRISPR-Cas9 cleavage in the chromosome bacteria is cell death. This can be conveniently used to eliminate specific genotypes from a mixed population bacteria, which achieved both vitro , e.g. select mutants, or vivo as an antimicrobial strategy. efficiency with Cas9 kills has been observed quite variable depending on target sequence, but little known about sequence determinants and mechanisms involved. Here we performed genome-wide screen E. coli...
Abstract Antibiotic resistance (AMR) in bacteria is a major threat to public health, and one of the key elements spread evolution AMR clinical pathogens transfer conjugative plasmids. The drivers have been extensively studied vitro , but plasmid-mediated vivo remains poorly explored. Here, we tracked clinically-relevant plasmid pOXA-48, which confers last-resort antibiotics carbapenems, large collection enterobacterial clones isolated from gut hospitalised patients. Combining genomic...
For the past 40 years, bacteriophage lambda has been crucial in revealing fundamental principles underlying control of transcription by elements positioned close to promoters. With discovery that CI repressors bound distant sites can interact efficiently, also provides a model for long range gene regulation, including action enhancer elements.
Abstract Over the past few years, tools that make use of Cas9 nuclease have led to many breakthroughs, including in control gene expression. The catalytically dead variant known as dCas9 can be guided by small RNAs block transcription target genes, a strategy also CRISPRi. Here, we reveal level complementarity between guide RNA and controls rate at which successfully blocks polymerase. We this mechanism precisely robustly reduce expression defined relative amounts. demonstrate broad...
Abstract In Gram-negative bacteria, coordinated remodelling of the outer membrane (OM) and peptidoglycan is crucial for envelope integrity. Envelope stress caused by unfolded OM proteins (OMPs) activates sigmaE (σ E ) in Enterobacteria. σ upregulates OMP biogenesis factors, including β-barrel assembly machinery (BAM) that catalyzes OMP-folding. Elevated activity, however, can be detrimental Here we report DolP (YraP), a -upregulated lipoprotein important integrity, novel interactor BAM...