The CRISPR-Cas gene editing system is being used in laboratory studies to block development of antimicrobial resistance (AMR). This work was carried out by researchers at the University of Exeter and the Institute of Hydrobiology in Dresden.
Their study appears in Microbiology today. The lead author is David Walker-Sünderhauf, of the University of Exeter.
AMR is a major global threat, with nearly five million deaths annually resulting from antibiotics failing to treat infection, according to the U.S. Centers for Disease Control and Prevention.
Walker-Sünderhauf said, “Antimicrobial resistance threatens to outstrip covid in terms of the number of global deaths. We urgently need new ways to stop resistance spreading between hosts. Our technology is showing early promise to eliminate resistance in a wide range of different bacteria.”
Bacteria often develop resistance when resistance-conferring genes are transported between hosts. One way that this occurs is via plasmids—circular strands of DNA, that can spread easily between bacteria, and swiftly replicate. This process can occur in people, and in environmental settings, such as waterways.
CRISPR is usually delivered using an engineered plasmid or bacteriophage. After uptake of the CRISPR delivery tool by recipient cells, the Cas9 nuclease cleaves a DNA sequence signaled by its single guide RNA (sgRNA). This leads to chromosome cleavage and cell death, or plasmid cleavage and resensitization to antibiotics.
The authors note that, “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.”
The Exeter team used the CRISPR-Cas gene editing system, which can target specific sequences of DNA, to engineer a plasmid that can specifically target the resistance gene for Gentamicin—a commonly used antibiotic. They engineered the broad host-range IncP1-plasmid pKJK5 (pKJK5::csg) to encode cas9 programmed to target an AMR gene.
In laboratory experiments, pKJK5::csg protected host cells from developing resistance. Furthermore, researchers found that the plasmid effectively targeted antimicrobial resistant genes in hosts to which it transferred, reversing their resistance.
The team reports that pKJK5::csg can block the uptake of AMR plasmids and 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.
Said Sünderhauf, “Our next step is to conduct experiments in more complex microbial communities. We hope one day it could be a way to reduce the spread of antimicrobial resistance in environments such as sewage treatment plants, which we know are breeding grounds for resistance.”
