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. 2020 Dec;59(1):e130.
doi: 10.1002/cpmc.130.

Programmable Gene Knockdown in Diverse Bacteria Using Mobile-CRISPRi

Amy B Banta  1   2 , Ryan D Ward  1   3 , Jennifer S Tran  1 , Emily E Bacon  1 , Jason M Peters  1   2   4   5
Affiliations

Programmable Gene Knockdown in Diverse Bacteria Using Mobile-CRISPRi

Amy B Banta et al. Curr Protoc Microbiol. 2020 Dec.

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.

Keywords: Bacillus subtilis; CRISPR-Cas9; CRISPRi; ESKAPE pathogens; Escherichia coli; Listeria monocytogenes; Zymomonas mobilis; biofuels; conjugation; functional genomics.

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Figures

Figure 1
Figure 1
CRISPRi and Mobile‐CRISPRi. (A) CRISPRi represses transcription by sterically blocking RNA polymerase (RNAP) elongation. (B) Mobile‐CRISPRi is a modular system containing sgRNAs and dcas9 that is inserted into a Tn7 vector or the ICEBs1 element for transfer to recipient bacteria. Spacers targeting new genes can be cloned into BsaI sites upstream of the sgRNA. (C) ICEBs1 Mobile‐CRISPRi transfers to recipient bacteria via biparental mating and integrates into the genome downstream of the leu2 tRNA gene. (D) Tn7 Mobile‐CRISPRi transfers to recipient bacteria via triparental mating with donors containing either a plasmid with Tn7 transposon ends flanking the CRISPRi components or a plasmid expressing Tn7 transposase genes. Tn7 integrates into the recipient genome downstream of the glmS gene.
Figure 2
Figure 2
sgRNA spacer cloning. Shown here is the sgRNA module from the Mobile‐CRISPRi plasmid pJMP1339. Annealed oligos with BsaI‐compatible sticky ends are ligated into the BsaI‐cut vector (BsaI recognition sites are lost in the cloning process). This figure depicts a spacer targeting mRFP, but 20‐nt spacer sequences targeting any gene of interest can be cloned using this protocol.
See this image and copyright information in PMC

References

Literature Cited

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Internet Resources
    1. https://docs.conda.io/projects/conda/en/latest/
    1. https://github.com/
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