Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism DOI Creative Commons
Jochem N. A. Vink, Koen J.A. Martens, Marnix Vlot

и другие.

Molecular Cell, Год журнала: 2019, Номер 77(1), С. 39 - 50.e10

Опубликована: Ноя. 14, 2019

Язык: Английский

Genome-wide gene expression tuning reveals diverse vulnerabilities of M. tuberculosis DOI Creative Commons
Barbara Bosch, Michael A. DeJesus, Nicholas C. Poulton

и другие.

Cell, Год журнала: 2021, Номер 184(17), С. 4579 - 4592.e24

Опубликована: Июль 22, 2021

Antibacterial agents target the products of essential genes but rarely achieve complete inhibition. Thus, all-or-none definition essentiality afforded by traditional genetic approaches fails to discern most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs. In contrast, gene "vulnerability" is a continuous, quantifiable trait that relates magnitude effect on fitness. We developed CRISPR interference-based functional genomics method systematically titrate expression Mycobacterium tuberculosis (Mtb) and monitor outcomes. identified highly vulnerable various processes, including novel targets unexplored for drug discovery. Equally important, we invulnerable genes, potentially explaining failed discovery efforts. Comparison vulnerability between reference hypervirulent Mtb isolate revealed conservation differential can predict antibacterial susceptibility. Our quantitatively redefine processes identify high-value development.

Язык: Английский

Процитировано

243

Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi DOI
Jason M. Peters, Byoung‐Mo Koo,

Ramiro Patino

и другие.

Nature Microbiology, Год журнала: 2018, Номер 4(2), С. 244 - 250

Опубликована: Дек. 20, 2018

Язык: Английский

Процитировано

234

Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors DOI Creative Commons
François Rousset, Lun Cui,

Elise Siouve

и другие.

PLoS Genetics, Год журнала: 2018, Номер 14(11), С. e1007749 - e1007749

Опубликована: Ноя. 7, 2018

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 could 79% previously reported essential and demonstrate non-essentiality some annotated essential. In addition, took advantage intermediate repression levels obtained when targeting template strand show that cells very sensitive expression level limited set genes. Our data visualized on CRISPRbrowser, custom web interface available at crispr.pasteur.fr. then apply discover required by phages λ, T4 186 kill their host, highlighting involvement diverse host pathways infection process three tested phages. colanic acid capsule synthesis shared resistance mechanism all Finally, plasmid packaging system transduction assay, for formation functional λ capsids, thus covering entire phage cycle. This study demonstrates usefulness convenience pooled bacteria paves way broader use tool bacterial genomics.

Язык: Английский

Процитировано

204

Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome DOI Creative Commons
Kathy N. Lam, Peter Spanogiannopoulos, Paola Soto-Perez

и другие.

Cell Reports, Год журнала: 2021, Номер 37(5), С. 109930 - 109930

Опубликована: Ноя. 1, 2021

Mechanistic insights into the role of human microbiome in predisposition to and treatment disease are limited by lack methods precisely add or remove microbial strains genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used deliver DNA Escherichia coli within mouse gastrointestinal (GI) tract. Delivery a programmable exogenous CRISPR-Cas9 system enables strain-specific depletion fluorescently marked isogenic during competitive colonization genomic deletions encompass target gene mice colonized with single strain. Multiple mechanisms allow E. escape targeting, including loss CRISPR array even entire system. These results provide robust experimentally tractable platform for editing, foundation refinement this approach increase targeting efficiency, proof concept extension other phage-bacterial pairs interest.

Язык: Английский

Процитировано

109

Bacillus subtilis cell diameter is determined by the opposing actions of two distinct cell wall synthetic systems DOI
Michael F. Dion, Mrinal Kapoor, Yingjie Sun

и другие.

Nature Microbiology, Год журнала: 2019, Номер 4(8), С. 1294 - 1305

Опубликована: Май 13, 2019

Язык: Английский

Процитировано

134

Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects DOI Creative Commons
A. Vigouroux, Baptiste Cordier, Andrey Aristov

и другие.

eLife, Год журнала: 2020, Номер 9

Опубликована: Янв. 6, 2020

Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped

Язык: Английский

Процитировано

124

Mismatch-CRISPRi Reveals the Co-varying Expression-Fitness Relationships of Essential Genes in Escherichia coli and Bacillus subtilis DOI Creative Commons

John S. Hawkins,

Melanie R. Silvis, Byoung‐Mo Koo

и другие.

Cell Systems, Год журнала: 2020, Номер 11(5), С. 523 - 535.e9

Опубликована: Окт. 19, 2020

Essential genes are the hubs of cellular networks, but lack high-throughput methods for titrating gene expression has limited our understanding fitness landscapes against which their levels optimized. We developed a modified CRISPRi system leveraging predictable reduction in efficacy imperfectly matched sgRNAs to generate defined activity and demonstrated its broad applicability. Using libraries mismatched predicted span full range knockdown levels, we characterized expression-fitness relationships most essential Escherichia coli Bacillus subtilis. find that these vary widely from linear bimodal similar within pathways. Notably, despite ∼2 billion years evolutionary separation between E. B. subtilis, homologs have with rare informative differences. Thus, may reflect homeostatic or constraints shared two organisms.

Язык: Английский

Процитировано

118

Tuning dCas9's ability to block transcription enables robust, noiseless knockdown of bacterial genes DOI Creative Commons
A. Vigouroux, Enno R. Oldewurtel, Lun Cui

и другие.

Molecular Systems Biology, Год журнала: 2018, Номер 14(3)

Опубликована: Март 1, 2018

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 Teeffelen orcid.org/0000-0002-0877-1294 Information Vigouroux1,2, Oldewurtel2, Cui1, *,1 *,2 1Synthetic 2Microbial *Corresponding author. Tel: +33 1 45 61 39 24; E-mail: 68 80 16; Molecular Systems (2018)14:e7899https://doi.org/10.15252/msb.20177899 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Over past few years, tools that make use Cas9 nuclease have led many breakthroughs, in control gene expression. The catalytically dead variant known as dCas9 can be guided small RNAs target genes, strategy also CRISPRi. Here, we reveal level complementarity between guide RNA controls rate at which polymerase “kicks out” from completes transcription. We mechanism precisely robustly reduce expression defined relative amounts. Alternatively, tuning repression changing concentration is noisy promoter-strength dependent. demonstrate broad applicability method study genetic regulation cellular physiology. First, characterize feedback strength model auto-repressor. Second, impact amount variations cell-wall synthesizing enzymes on cell morphology. Finally, multiplex system obtain any combination fractional two genes. Synopsis When encounters inactivated nuclease, it has certain probability going through depending sequence. This property exploited multiple their native locus. For high enough concentration, locus saturated only depends RNAP passage probability. Imperfect allows fine-tuning consequently levels. In saturating conditions, does not produce additional noise applied measure response circuit, analyze how synthesis affect shape, tune levels independently. Introduction A powerful way investigate bacteria vary cell. To end, are typically placed under inducible promoters. While easy implement, approach disadvantages: lie outside dynamic range promoter. promoters increase comparison with (Elowitz et al, 2002). And third, orthogonal systems exist, thus making multiplexing difficult. Recently, different strategies been devised knock down amounts levels: Specifically, antisense manner (Brophy Voigt, 2016). works well moderate promoter strength, becomes less efficient stronger As an alternative strategy, knocked varying degrees using CRISPR technology (Bikard 2013; Qi 2013). catalytic mutant form RNA-guided Streptococcus pyogenes (dCas9) easily programmed bind position interest chromosome, requirement “NGG” protospacer adjacent motif (PAM). unable cleave DNA, but still binds DNA strongly. If chosen downstream promoter, serves roadblock blocks elongation. single-cell level, interesting implications immune system. then develop precise noise-preserving independent strength. Target search begins probing presence PAM followed melting complementarity-dependent strand invasion (Sternberg 2014; Szczelkun 2014). PAM-proximal region seed sequence important binding, several mismatches PAM-distal tolerated demonstrated binding assays (Kuscu Wu 2014) monitoring target-gene Eschericha coli degree controlled quantitatively ways: first, impacts DNA. recently Bacillus subtilis where was xylose-inducible (Peters 2016), E. strain modified enable tunable PBAD (Li 2016); second, introducing perfectly matched leads very strong repression, decreasing progressively reduces compare these characterizing properties dCas9-mediated level. us propose novel physical repression. It previously assumed decreased would decrease virtue reduced occupancy (Farasat Salis, mechanism: inside open reading frame (ORF), determines (RNAP) kicks out during attempt, while spontaneous unbinding negligibly small. saturate target, alone desirable properties: add extrinsic On contrary, inherently target. complementarity-based fluorescent-protein reporters inserted upstream its reporter fusions rather than direct targeting yields predictable fold characterized provides monitor single cells. versatility our examples: accurate quantify auto-repressor measuring much actual differs rate. take advantage steady-state growth operon coding essential “rod” complex, PBP2 RodA. multiplexed Results Varying RNA-target controlling without addition CRISPR-dCas9 modulates integrated cassettes constitutively expressed reporters, sfgfp superfolder green fluorescent protein (GFP) mCherry red (RFP) chromosomal loci MG1655. repress either knock-down, dcas9 S. Ptet anhydrotetracycline (aTc) (Qi GFP- RFP-coding ORFs array necessary tracrRNA, complex together (Hsu Inducing setup did (Appendix Fig S1). measured stability over time saw 5 days culture. Once stopped induction all 40 clones tested recovered stable, show toxicity. system, tuned modulating aTc concentrations or spacer numbers 5′ side spacer. employed GFP high-throughput microscopy (Fig 1A B). expected, average increasing complementarity. However, distributions differed significantly modes modulation 1C D). large cell-to-cell fluctuations intermediate regime, strongly 1D Appendix S2). induced, non-repressed constitutive were recovered. expression, presumably site elaborated below. inducing constant 100 ng/ml maintained (standard deviation mean) almost plateau value about 0.3 (corresponding 30%) similar measurements made others wild-type (Taniguchi 2010). Complementarity-based qualitatively transcriptional repressors. example, Lac repressor part targets fivefold compared unrepressed case (see S3), agreement previous Accordingly, observed if modulated inducer concentration. proposed here precision Figure 1. modulate generating A, B. Average obtained (A) (B) inducer. Relative given relatively non-targeting Individual points represent replicates. Horizontal bars median three C, D. Distribution each experiment panels (A Curves same color replicates condition. Mechanistic dCas9-targeted product probabilities: P(stop) blocking upon collision occupying (termed occupancy). determined kon, [dCas9], kout. kout, turn, sum transcription-independent kick-out due 4 details). F. schematically illustrate behavior (left) (right), respectively. data available online figure. Data [msb177899-sup-0002-SDataFig1.zip] Download figure PowerPoint transcribe dCas9-bound lack suggested might test hypothesis, fraction active complexes roughly factor decoy 2A). population-averaged flow cytometry. Indeed, found absence both low 2B), confirming hypothesis 20 11 bp. remained true even spacers array, regardless S4). effectiveness confirmed gradually lowering until transition no happened higher decoys one S4B), complex. cases, induction, residual reached around 3%, corresponding concentration-independent regime. note following population averages performed cytometry generally noisier results presented 2. mismatched depend Left: Schematic assay used dependence R20 RFP perfect match. R11 bp C Introducing acts halves Right: Northern blot measurement processed R20, reflecting carrying moment measurement. Error standard deviations biological a.u.: arbitrary units. Flow levels, point representing replicate. values normalized respect (C-C). Expression differ (C-R20 vs. R20-R20, P-value: 0.68), nor when order reversed R20-C, 0.21), (C-R11 R11-R11, 0.53). P-values come two-sided Student's t-test natural logarithms mean (significance threshold: 0.017 after Bonferroni correction). 2 [msb177899-sup-0003-SDataFig2.zip] Previously, thought solely is, rates unbinding. According simple view, should lead equilibrium occupancy, fully occupied. view clear contradiction independence 2B). suggests dCas9, bound times, must responsible reconcile robustness hypothesize possible suggest ≤ 1E). = 1, efficiently every collide. At opposite extreme, 0, never 1F). mechanism, γ0 P(bound) Therefore, close saturated. longer explaining Interestingly, moved ORF region, increased S5). finding pass occupied thanks processive activity, cannot relies diffusion. know whether influence definition, Repression measurably affected strengths, verify prediction, put strengths (P127 PPhlf) blocked four number mismatches. PPhlF times P127 3A), regard promoter's initial identical 3B). 12 weaker PLac mM IPTG S6). These observations confirm conditions 3. conditionsRelative repressed set (A, B) non-saturating (C) concentrations. Raw P127, C. Experimental predicted (using lower aTc). PPhlf up six RNA, quantitative Appendix). bars: error computational prediction. 3 [msb177899-sup-0004-SDataFig3.zip] supporting “kick-out” ejection successful wondered ejected events. displacements they could RNAP, kon[dCas9] kout combined RNAP-induced ejections, possibly replication-fork-based (Jones 2017; see therefore P(bound), which, fold. observation agrees full (14 bp) complementarity, respectively 3C S7A). predicts dominated events, rare (4; S7B). compatible leaves spontaneously gets kicked replication. prediction consistent long half-life reported vivo 2017) vitro expected valid 14 bp, 1A) remains become equally collision-based ejections below some Yet, applications below, rebinding paragraph). proportional rate, principle events reads template increases temperature (Wiktor 2016) 42°C, suggesting temperature. bears possibility robust dCas9-copy temperature, condition saturation fulfilled. robustness, temperatures ranging 30 42°C. decayed continuously 4), displaying sharp 37 Regardless S8). From model, conclude being RNAP. indicates work independently tested. 4. efficiency temperaturesRelative temperatures. [msb177899-sup-0005-SDataFig4.zip] insertions contexts Precision, versatile strategy. context, insert translational gene. provide convenient CRISPR-based perform inspired allelic exchange (Pósfai 1999) Text S9). library plasmids introduced desired sequences. taking established above. Furthermore, revealing variations. addgene (https://www.addgene.org/depositor-collections/bikard-crispr-repression/). following, physiology: effect morphology growth. uncover circuits, chose described PhlF auto-repres

Язык: Английский

Процитировано

113

Visualisation of dCas9 target search in vivo using an open-microscopy framework DOI Creative Commons
Koen J.A. Martens, Sam P. B. van Beljouw,

Simon van der

и другие.

Nature Communications, Год журнала: 2019, Номер 10(1)

Опубликована: Авг. 7, 2019

CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to cellular concentration Cas9 have remained elusive. Effective requires constant screening protospacer adjacent motif (PAM) a 30 ms upper limit for was recently found. To further quantify rapid switching between DNA-bound freely-diffusing states dCas9, we developed an open-microscopy framework, miCube, introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our reveals that dCas9 PAMs 40% time Gram-positive Lactoccous lactis, averaging 17 ± 4 per binding event. Using heterogeneous expression, determine number target-containing plasmids derive copy dependent cleavage. Furthermore, show not irreversibly bound sites can still interfere with plasmid replication. Taken together, our quantitative data facilitates optimization CRISPR-Cas toolbox.

Язык: Английский

Процитировано

107

Editing the microbiome the CRISPR way DOI Creative Commons

Gayetri Ramachandran,

David Bikard

Philosophical Transactions of the Royal Society B Biological Sciences, Год журнала: 2019, Номер 374(1772), С. 20180103 - 20180103

Опубликована: Март 25, 2019

Our bodies are colonized by a complex ecosystem of bacteria, unicellular eukaryotes and their viruses that together play major role in our health. Over the past few years tools derived from prokaryotic immune system known as CRISPR-Cas have empowered researchers to modify study organisms with unprecedented ease efficiency. Here we discuss how various types systems can be used genome gut microorganisms bacteriophages. also delivered bacterial population programmed specifically eliminate members microbiome. Finally, engineered control gene expression modulate production metabolites proteins. Together these provide exciting opportunities investigate interplay between microbiome bodies, present new avenues for development drugs target This article is part discussion meeting issue ‘The ecology evolution adaptive systems’.

Язык: Английский

Процитировано

103