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Programmable Gene Knockdown in Diverse Bacteria Using Mobile-CRISPRi.

Amy B Banta1,2, Ryan D Ward1,3, Jennifer S Tran1

  • 1Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin.

Current Protocols in Microbiology
|December 17, 2020
PubMed
Summary
This summary is machine-generated.

We provide protocols for Mobile-CRISPRi, a tool for bacterial gene knockdown. This system enables targeted gene silencing in diverse bacteria using CRISPR interference (CRISPRi) for functional genomics research.

Keywords:
Bacillus subtilisCRISPR-Cas9CRISPRiESKAPE pathogensEscherichia coliListeria monocytogenesZymomonas mobilisbiofuelsconjugationfunctional genomics

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Bacterial genome sequencing has revealed numerous novel genes requiring functional characterization.
  • Effective genetic tools are essential for exploring these genes across diverse bacterial species.
  • CRISPR interference (CRISPRi) offers a programmable method for gene knockdown using a guide RNA and a dCas9 nuclease.

Purpose of the Study:

  • To provide detailed protocols for modifying and transferring Mobile-CRISPRi vectors.
  • To enable targeted gene knockdown in bacteria of interest.
  • To offer strategies for optimizing CRISPRi knockdown, transfer, and integration.

Main Methods:

  • sgRNA design and cloning into Mobile-CRISPRi vectors.
  • Tn7-based transfer of Mobile-CRISPRi to Gram-negative bacteria.
  • ICEBs1-mediated transfer of Mobile-CRISPRi to Bacillales.

Main Results:

  • Detailed protocols for Mobile-CRISPRi vector modification and transfer are presented.
  • Strategies for optimizing CRISPRi knockdown efficiency and system integration are discussed.
  • The study covers essential steps from sgRNA design to bacterial transformation.

Conclusions:

  • Mobile-CRISPRi provides a versatile platform for functional genomics in diverse bacteria.
  • The provided protocols facilitate the application of CRISPRi for targeted gene knockdown.
  • This work enhances the utility of CRISPRi tools for exploring bacterial gene function.