Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • David Walker-Sünderhauf - , University of Exeter (Author)
  • Uli Klümper - , Institute of Hydrobiology, Chair of Limnology (Author)
  • Elizabeth Pursey - , University of Exeter (Author)
  • Edze R Westra - , University of Exeter (Author)
  • William H Gaze - , University of Exeter (Author)
  • Stineke van Houte - , University of Exeter (Author)

Abstract

Antimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode cas9 programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from Escherichia coli. Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of Pseudomonas. This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.

Details

Original languageEnglish
Article number001334
JournalMicrobiology
Volume169
Issue number5
Publication statusPublished - May 2023
Peer-reviewedYes

External IDs

Scopus 85160125038
ORCID /0000-0002-4169-6548/work/142247386

Keywords

Keywords

  • Humans, Animals, Swine, CRISPR-Cas Systems, Host Specificity, Bacteriophages, Biological Transport, Escherichia coli/genetics, Plasmids/genetics

Library keywords