High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae.
Xue Liu,Xue Liu,Clement Gallay,Morten Kjos,Morten Kjos,Arnau Domenech,Jelle Slager,Sebastiaan P van Kessel,Kèvin Knoops,Robin A. Sorg,Jing-Ren Zhang,Jan-Willem Veening,Jan-Willem Veening +12 more
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TLDR
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.read more
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Journal ArticleDOI
A decade of advances in transposon-insertion sequencing
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.
Journal ArticleDOI
A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9
Lun Cui,Antoine Vigouroux,François Rousset,François Rousset,Hugo Varet,Varun Khanna,David Bikard +6 more
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.
Journal ArticleDOI
Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors.
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.
Journal ArticleDOI
Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi.
Jason M. Peters,Jason M. Peters,Byoung-Mo Koo,Ramiro Patino,Gary E. Heussler,Cameron C. Hearne,Jiuxin Qu,Yuki F. Inclan,John S. Hawkins,Candy H. S. Lu,Melanie R. Silvis,M. Michael Harden,Hendrik Osadnik,Joseph E. Peters,Joanne N. Engel,Rachel J. Dutton,Alan D. Grossman,Carol A. Gross,Oren S. Rosenberg +18 more
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.
Journal ArticleDOI
Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi.
Henry H. Lee,Nili Ostrov,Brandon G. Wong,Michaela A. Gold,Ahmad S. Khalil,Ahmad S. Khalil,George M. Church,George M. Church +7 more
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.
References
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Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms.
TL;DR: Fitness for each gene of Streptococcus pneumoniae, a causative agent of pneumonia and meningitis is determined using a method based on the assembly of a saturated Mariner transposon insertion library.
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