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

## TL;DR

A new CRISPRi system for Clostridioides difficile was developed that doesn't need antibiotics to maintain gene knockdown.

## Contribution

A two-plasmid CRISPRi system for C. difficile was created without requiring antibiotic selection.

## Key findings

- The system uses an optimized tetracycline-inducible dCas9 for gene knockdown.
- A toxin-antitoxin system ensures plasmid stability without antibiotics.
- Successful knockdown of the essential gene walA was demonstrated.

## 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.