CRICON: Conjugative Delivery of CRISPR-Cas9 System for Eradicating Antibiotic Resistance (CRICON)


Main funder


Funds granted by main funder (€)

255 000,00


Funding program

Foundation (Funding programs)


Project timetable

Project start date: 01/08/2019

Project end date: 31/08/2022


Summary

In vivo utilization of CRISPR genetic editing requires the introduction of editing components to targeted cells. The currently developed state of the art of in vivo delivery of antibiotic-resistant gene deleting CRISPR systems to bacteria exploits modified bacteriophages (viruses that infect bacteria). These phage-based delivery systems have acquired major funding and attention in spite of their inevitable shortcomings: phages always have a relatively small host range, due to specific receptor binding, and bacteria readily become resistant to phage infections.
In the proposed project, I aim to establish an alternative (and preliminarily tested) approach, namely bacterial conjugation, to deliver CRISPR editing components to antibiotic-resistant bacterial communities. This system has several advantages over phage delivery: it adapts to natural bacterial communities by being transferred from the initial donors to resident flora, it is not dependent on any particular receptor(s), it is immune to host defences (such as restriction-modification systems) due to synthesis of the complementary strand in the recipient cell, and it can target multiple resistance gene variants simultaneously from the engineered Cas9 guiding spacer array. The developed system provides long-term sustained activity against antibiotic-resistant bacteria compared to the burst-like activity of phage-based applications.
The project includes several steps: optimization of CRICON (CRISPR via conjugation) to deliver a probiotic bacterial cocktail, engineering of efficient CRICON plasmids, in vitro tests to evaluate the efficiency and potential obstacles in resistance clearance on molecular and community levels, in vivo experiments with a rodent ESBL-carriage model, and modelling of CRICON-based genetic modification of heterogeneous bacterial communities. In the future, the CRICON system can be used in various types of studies seeking to introduce or delete genes from natural bacterial populations.


Principal Investigator


Primary responsible unit


Last updated on 2020-23-10 at 11:30