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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Directed Evolution of CRISPR/Cas Systems for Precise Gene Editing.
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Gene Silencing Through CRISPR Interference in Bacteria: Current Advances and Future Prospects
TL;DR: In this article, the authors summarized the characteristics of the CRISPRi system, such as programmable, highly efficient, and specific, and demonstrated its applications in functional genetic screening and highlighted its potential to dissect the underlying mechanism of pathogenesis.
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Phage proteins block and trigger retron toxin/antitoxin systems
Jacob Bobonis,Karin Mitosch,André Mateus,George Kritikos,Elfenbein,Mikhail M. Savitski,Helene Andrews-Polymenis,Athanasios Typas +7 more
TL;DR: It is proposed that retron-TAs act as abortive-infection anti-phage defense systems, and delineate mechanistic principles by which phages trigger or block them.
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Genome-Wide CRISPRi-Based Identification of Targets for Decoupling Growth from Production.
Songyuan Li,Christian Bille Jendresen,Jenny Marie Landberg,Lasse Ebdrup Pedersen,Nikolaus Sonnenschein,Sheila Ingemann Jensen,Alex Toftgaard Nielsen +6 more
TL;DR: A genome-wide CRISPRi screen is conducted to discover growth switches suitable for decoupling growth and production in Escherichia coli, and the approach can be applied to improve bio-based production in other microorganisms.
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