High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae.
TL;DR: The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.
Abstract: Genome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn-seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high-content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi-based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp-proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP-dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.
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TL;DR: The recent applications of TIS to answer overarching biological questions are discussed and emerging and multidisciplinary methods that build on TIS are explored, with an eye towards future applications.
Abstract: It has been 10 years since the introduction of modern transposon-insertion sequencing (TIS) methods, which combine genome-wide transposon mutagenesis with high-throughput sequencing to estimate the fitness contribution or essentiality of each genetic component in a bacterial genome. Four TIS variations were published in 2009: transposon sequencing (Tn-Seq), transposon-directed insertion site sequencing (TraDIS), insertion sequencing (INSeq) and high-throughput insertion tracking by deep sequencing (HITS). TIS has since become an important tool for molecular microbiologists, being one of the few genome-wide techniques that directly links phenotype to genotype and ultimately can assign gene function. In this Review, we discuss the recent applications of TIS to answer overarching biological questions. We explore emerging and multidisciplinary methods that build on TIS, with an eye towards future applications.
177 citations
TL;DR: It is revealed that guide RNAs sharing specific 5-nucleotide seed sequences can produce strong fitness defects or even kill E. coli regardless of the other 15 nucleotides of guide sequence, and design rules to minimize off-target effects are provided.
Abstract: 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 to direct the catalytically dead Cas9 (dCas9) to block gene transcription in Escherichia coli. Using a machine-learning approach, we reveal that guide RNAs sharing specific 5-nucleotide seed sequences can produce strong fitness defects or even kill E. coli regardless of the other 15 nucleotides of guide sequence. This effect occurs at high dCas9 concentrations and can be alleviated by tuning the expression of dCas9 while maintaining strong on-target repression. Our results also highlight the fact that off-targets with as little as nine nucleotides of homology to the guide RNA can strongly block gene expression. Altogether this study provides important design rules to safely use dCas9 in E. coli.
171 citations
TL;DR: This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria and paves the way for their broader use as a powerful tool in bacterial genomics.
Abstract: High-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a starting pool of ~ 92,000 sgRNAs which target random positions in the chromosome of E. coli. To benchmark our method, we first investigate its utility to predict gene essentiality in the genome of E. coli during growth in rich medium. We could identify 79% of the genes previously reported as essential and demonstrate the non-essentiality of some genes annotated as essential. In addition, we took advantage of the intermediate repression levels obtained when targeting the template strand of genes to show that cells are very sensitive to the expression level of a limited set of essential genes. Our data can be visualized on CRISPRbrowser, a custom web interface available at crispr.pasteur.fr. We then apply the screen to discover E. coli genes required by phages λ, T4 and 186 to kill their host, highlighting the involvement of diverse host pathways in the infection process of the three tested phages. We also identify colanic acid capsule synthesis as a shared resistance mechanism to all three phages. Finally, using a plasmid packaging system and a transduction assay, we identify genes required for the formation of functional λ capsids, thus covering the entire phage cycle. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria and paves the way for their broader use as a powerful tool in bacterial genomics.
141 citations
TL;DR: ‘Mobile-CRISpri’ is established, a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation that enables genetic investigations in non-model bacteria.
Abstract: The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans1-6. However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.
135 citations
TL;DR: A genome-wide CRISPR interference screen of the fast-growing Vibrio natriegens bacterium elucidates the make-up of minimal genomes and the metabolic pathways enabling rapid bacterial replication.
Abstract: The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time1,2 and metabolic prowess3,4. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens. A genome-wide CRISPR interference screen of the fast-growing Vibrio natriegens bacterium elucidates the make-up of minimal genomes and the metabolic pathways enabling rapid bacterial replication.
122 citations
References
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TL;DR: This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale and can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects.
4,282 citations
"High-throughput CRISPRi phenotyping..." refers background or methods in this paper
...When targeting the non-template strand of a gene by complementary basepairing of the sgRNA with the target DNA, the dCas9-sgRNA-DNA complex acts as a roadblock for RNA polymerase (RNAP) and thereby represses transcription of the target genes (Qi et al, 2013; Peters et al, 2016) (Fig 1A)....
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...We chose the guide sequences as close as possible to the 50 end of the coding sequence of the targeted gene (Qi et al, 2013)....
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...It was shown that fabT and fabH are cotranscribed (Lu & Rock, 2006), but the transcription pattern of the other genes is still unknown, which makes functional study of these genes with CRISPRi very difficult due to polar effects of the block of transcription elongation (Qi et al, 2013)....
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...CRISPRi is based on expression of a nuclease-inactive Streptococcus pyogenes Cas9 (dCas9), which together with expression of a single-guide RNA (sgRNA) targets the gene of interest (Bikard et al, 2013; Qi et al, 2013; Peters et al, 2016)....
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...in S. pneumoniae Streptococcus pyogenes dcas9 (dcas9sp) was obtained from Addgene (Addgene #44249, Qi et al, 2013) and subcloned into plasmid pJWV102 (Veening laboratory collection) with the IPTG-inducible promoter Plac (Sorg, 2016) replacing PZn, resulting in plasmid pJWV102-Plac-dcas9sp.…...
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TL;DR: In this article, exact dynamic programming algorithms can be used to compute ground states, base pairing probabilities, as well as thermodynamic properties of nucleic acids based on carefully measured thermodynamic parameters.
Abstract: Background
Secondary structure forms an important intermediate level of description of nucleic acids that encapsulates the dominating part of the folding energy, is often well conserved in evolution, and is routinely used as a basis to explain experimental findings. Based on carefully measured thermodynamic parameters, exact dynamic programming algorithms can be used to compute ground states, base pairing probabilities, as well as thermodynamic properties.
3,620 citations
TL;DR: A reprint of Avery, MacLeod, and McCarty's article on the 35th anniversary of its original publication.
Abstract: 1. From Type III pneumococci a biologically active fraction has been isolated in highly purified form which in exceedingly minute amounts is capable under appropriate cultural conditions of inducing the transformation of unencapsulated R variants of Pneumococcus Type II into fully encapsulated cells of the same specific type as that of the heat-killed microorganisms from which the inducing material was recovered. 2. Methods for the isolation and purification of the active transforming material are described. 3. The data obtained by chemical, enzymatic, and serological analyses together with the results of preliminary studies by electrophoresis, ultracentrifugation, and ultraviolet spectroscopy indicate that, within the limits of the methods, the active fraction contains no demonstrable protein, unbound lipid, or serologically reactive polysaccharide and consists principally, if not solely, of a highly polymerized, viscous form of desoxyribonucleic acid. 4. Evidence is presented that the chemically induced alterations in cellular structure and function are predictable, type-specific, and transmissible in series. The various hypotheses that have been advanced concerning the nature of these changes are reviewed.
2,953 citations
"High-throughput CRISPRi phenotyping..." refers methods in this paper
...Using Tn-seq and CRISPRi, we refined the list of genes that are either essential for viability or for fitness in S. pneumoniae strain D39 (Avery et al, 1944)....
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TL;DR: Plasmid cloning vectors that enable insertion of DNA fragments between the inducible ara (arabinose) promoter and the lac (lactose) structural genes have been constructed and used for the detection and analysis of signals that control gene transcription.
2,442 citations
TL;DR: The feasibility of IPCR is shown by amplifying the sequences that flank an IS1 element in the genome of a natural isolate of Escherichia coli.
Abstract: A method is presented for the rapid in vitro amplification of DNA sequences that flank a region of known sequence. The method uses the polymerase chain reaction (PCR), but it has the primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle. This procedure of inverse PCR (IPCR) has many applications in molecular genetics, for example, the amplification and identification of sequences flanking transposable elements. In this paper we show the feasibility of IPCR by amplifying the sequences that flank an IS1 element in the genome of a natural isolate of Escherichia coli.
1,953 citations
"High-throughput CRISPRi phenotyping..." refers methods in this paper
...To test whether S. pneumoniae YabA is also a negative regulator of initiation of DNA replication, we determined the oriC-ter ratio using real-time quantitative PCR (qPCR)....
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...We found that 79% of the transformants produced by infusion cloning had a growth defect upon dCas9 induction with IPTG (38 out of 48 colonies), whereas 26% of the transformants generated by inverse PCR A C D B Figure 1....
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...With inverse PCR, we used a phosphorylated universal primer, together with a gene-specific primer to fuse the 20-nt basepairing region into the vector by PCR, followed by blunt-end ligation and direct transformation into S. pneumoniae D39 strain DCI23....
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...Based on the sgRNAluc plasmid (Fig 2A), we tested two different cloning strategies to introduce the unique 20-nt base-pairing region for each gene: infusion cloning and inverse PCR (Ochman et al, 1988; Irwin et al, 2012; Larson et al, 2013) (Fig EV2A)....
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...The pPEPX-P3-sgRNAluc is used as the template for generation of other sgRNAs by infusion cloning or by the inverse PCR method....
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