Dataset Information

Programmable Gene Knockdown in Diverse Bacteria Using Mobile-CRISPRi.

ABSTRACT: Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene-knockdown tool that uses an RNA-protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile-CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile-CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile-CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile-CRISPRi vectors, Tn7 transfer of Mobile-CRISPRi to Gram-negative bacteria, and ICEBs1 transfer of Mobile-CRISPRi to Bacillales. © 2020 The Authors. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning of new sgRNA spacers into Mobile-CRISPRi vectors Basic Protocol 3: Tn7 transfer of Mobile-CRISPRi to Gram-negative bacteria Basic Protocol 4: ICEBs1 transfer of Mobile-CRISPRi to Bacillales Support Protocol 1: Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2: Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3: Transformation of E. coli by electroporation Support Protocol 4: Transformation of CaCl₂ -competent E. coli.

SUBMITTER: Banta AB

PROVIDER: S-EPMC7809906 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models.IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

Project description:CRISPR interference (CRISPRi) has facilitated the study of essential genes in diverse organisms using both high-throughput and targeted approaches. Despite the promise of this technique, no comprehensive arrayed CRISPRi library targeting essential genes exists for the model bacterium Escherichia coli, or for any Gram-negative species. Here, we built and characterized such a library. Each of the ∼500 strains in our E. coli library contains an inducible, chromosomally integrated single guide RNA (sgRNA) targeting an essential (or selected nonessential) gene and can be mated with a pseudo-Hfr donor strain carrying a dcas9 cassette to create a CRISPRi knockdown strain. Using this system, we built an arrayed library of CRISPRi strains and performed population and single-cell growth and morphology measurements as well as targeted follow-up experiments. These studies found that inhibiting translation causes an extended lag phase, identified new modulators of cell morphology, and revealed that the morphogene mreB is subject to transcriptional feedback regulation, which is critical for the maintenance of morphology. Our findings highlight canonical and noncanonical roles for essential genes in numerous aspects of cellular homeostasis. IMPORTANCE Essential genes make up only ∼5 to 10% of the genetic complement in most organisms but occupy much of their protein synthesis and account for almost all antibiotic targets. Despite the importance of essential genes, their intractability has, until recently, hampered efforts to study them. CRISPRi has facilitated the study of essential genes by allowing inducible and titratable depletion. However, all large-scale CRISPRi studies in Gram-negative bacteria thus far have used plasmids to express CRISPRi components and have been constructed in pools, limiting their utility for targeted assays and complicating the determination of antibiotic effects. Here, we use a modular method to construct an arrayed library of chromosomally integrated CRISPRi strains targeting the essential genes of the model bacterium Escherichia coli. This library enables targeted studies of essential gene depletions and high-throughput determination of antibiotic targets and facilitates studies targeting the outer membrane, an essential component that serves as the major barrier to antibiotics.

Dataset Information

Programmable Gene Knockdown in Diverse Bacteria Using Mobile-CRISPRi.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets