Plasmid sequences and availability of a two-plasmid system for CRISPRi knockdown of Clostridioides difficile genes without antibiotic selection

Michelle Chua; Daniel Erickson; James Collins

doi:10.1128/mra.00116-25

Plasmid sequences and availability of a two-plasmid system for CRISPRi knockdown of Clostridioides difficile genes without antibiotic selection

Microbiol Resour Announc. 2025 Jun 12;14(6):e0011625. doi: 10.1128/mra.00116-25. Epub 2025 Apr 28.

Authors

Michelle Chua^{1

2}, Daniel Erickson^{1

2}, James Collins^{1

2

3}

Affiliations

¹ Department of Microbiology & Immunology, University of Louisville, Louisville, Kentucky, USA.
² Center for Predictive Medicine, University of Louisville, Louisville, Kentucky, USA.
³ Center for Microbiomics, Inflammation and Pathogenicity, University of Louisville, Louisville, Kentucky, USA.

Abstract

A two-plasmid CRISPRi system for Clostridioides difficile that does not require antibiotic maintenance was developed. pJAK184.tetR.PT5-3.dCas9 contains an optimized tetracycline-inducible dCas9 for chromosomal insertion. pJC.15A.sgRNA.TA encodes a toxin-antitoxin system for stable maintenance, and mCherry, which is exchangeable for a customized sgRNA. We demonstrate the knockdown of the essential gene walA.

Keywords: CRISPRi; Clostridioides difficile; antibiotic-free maintenance; dCas9.

Abstract

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