ArticleCurrent protocols in microbiology2020
Programmable Gene Knockdown in Diverse Bacteria Using Mobile-CRISPRi.
Article in Current protocols in microbiology, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 33 papers.
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Who cites it
33 citing papers in PubMed, 51 citations in OpenAlex.
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- CRISPR-Cas9: Genome Engineering and Future Vaccine Applications.Molecular biotechnology · 2026Review
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- State-dependent promoter switching mediates integrase gene transcription in the integrative and conjugative element ICEKp1 of Klebsiella pneumoniae.Nucleic acids research · 2026Article
- Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogenApplied and environmental microbiology · 2026Article
- Orthogonal chemical genomics approaches reveal genomic targets for increasing anaerobic chemical tolerance inmSystems · 2026Article
- Transforming RNA-Based Gene Therapy with Innovative Nanocarriers for siRNA and miRNA Delivery.Current gene therapy · 2026Review
- Systematic genome-wide mapping of host determinants of bacteriophage infectivity.Cell systems · 2025Article
- A programmable, selection-free CRISPR interference system iniScience · 2025Article
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- Generation and validation of a versatile inducible multiplex CRISPRi system to examine bacterial regulation in the Euprymna-Vibrio fischeri symbiosis.Archives of microbiology · 2025Article
- Chemical genomics informs antibiotic and essential gene function in Acinetobacter baumannii.PLoS genetics · 2025Article
- Co-zorbs: Motile, multispecies biofilms aid transport of diverse bacterial species.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- GLiDe: a web-based genome-scale CRISPRi sgRNA design tool for prokaryotes.BMC bioinformatics · 2025Article
- Characterization of halotolerant Kushneria isolates that stimulate growth of alfalfa in saline conditions.PloS one · 2025Article
- Programming CRISPRi to control the lifecycle of bacteriophage T7.Frontiers in microbiology · 2025Article
- A High-Throughput Droplet-based Method to Facilitate Microbial Conjugation.Bio-protocol · 2024Article
- It's complicated: relationships between integrative and conjugative elements and their bacterial hosts.Current opinion in microbiology · 2024Review
Corrections and comments
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
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 CaCl
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.