High-contrast imaging of cellular non-repetitive drug-resistant genes via in situ dead Cas12a-labeled PCR

Chem Commun (Camb). 2024 Sep 20;60(76):10524-10527. doi: 10.1039/d4cc03059a.

Abstract

In situ imaging of genes of pathogenic bacteria can profile cellular heterogeneity, such as the emergence of drug resistance. Fluorescence in situ hybridization (FISH) serves as a classic approach to image mRNAs inside cells, but it remains challenging to elucidate genomic DNAs and relies on multiple fluorescently labeled probes. Herein, we present a dead Cas12a (dCas12a)-labeled polymerase chain reaction (CasPCR) assay for high-contrast imaging of cellular drug-resistant genes. We employed a syncretic dCas12a-green fluorescent protein (dCas12a-GFP) to tag the amplicons, thereby enabling high-contrast imaging and avoiding multiple fluorescently labeled probes. The CasPCR assay can quantify quinolone-resistant Salmonella enterica in mixed populations and identify them isolated from poultry farms.

MeSH terms

  • Animals
  • Anti-Bacterial Agents / chemistry
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems / genetics
  • Drug Resistance, Bacterial / genetics
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • In Situ Hybridization, Fluorescence / methods
  • Polymerase Chain Reaction*
  • Quinolones / pharmacology
  • Salmonella enterica* / genetics

Substances

  • Bacterial Proteins
  • Cas12a protein
  • CRISPR-Associated Proteins
  • Endodeoxyribonucleases
  • Green Fluorescent Proteins
  • Quinolones
  • Anti-Bacterial Agents