Award details

CRISPR-Cas9 gene drives to fight antimicrobial resistance

Reference	BB/R010781/1
Principal Investigator / Supervisor	Dr Stineke van Houte
Co-Investigators / Co-Supervisors
Institution	University of Exeter
Department	Biosciences
Funding type	Research
Value (£)	304,164
Status	Completed
Type	Fellowships
Start date	01/01/2018
End date	03/09/2021
Duration	44 months

Abstract

Antimicrobial resistance (AMR) is one of the greatest threats of our time. Recently it was shown that CRISPR-Cas9 can be used to eradicate AMR genes from bacterial populations in the lab. I aim to take the development of this technology to the next level by testing for the first time whether CRISPR-Cas9 can eradicate AMR from a complex microbial community. First, I will design synthetic gene drive elements, based on a combination of conjugation, transposition and recombination, to "drive" AMR-targeting CRISPR-Cas9 genes through a pig gut microbial community, using a conjugative plasmid with extraordinarily broad host range as delivery vehicle. Second, I will measure the fitness costs of the different CRISPR-Cas9 designs, and analyze their ability to drive through the community. These data will feed into a theoretical model to predict the optimal parameters for successful spread of CRISPR-Cas9. I will then monitor the effectiveness of the most promising CRISPR-Cas9 design in removing AMR, both from the pig gut community and from a simple derived community. Third, I will use state-of-the-art molecular techniques, including metagenomics, qPCR and epicPCR, to also measure the ecological and evolutionary consequences of AMR removal, such as increased frequencies of other AMR genes and evolution of resistance against CRISPR-Cas9 targeting. The powerful combination of observing these consequences in a "real" complex community, and performing more controlled experiments with simpler derived communities, will allow me to tease apart the mechanisms underlying the observed changes. In parallel, I will develop theory to generate specific predictions on the consequences of AMR removal. I will subsequently test these predictions in my empirical system, allowing for maximum synergy between theory and empirical data. This ambitious and innovative proof-of-concept study will be of major importance in reducing the impact of AMR on animal and human health.

Summary

Antimicrobial resistance (AMR) is one of the greatest threats of our time causing a predicted 10 million human deaths per year by 2050 with a total cost of $100 trillion. Recently, a revolutionary technology has been developed, known as CRISPR-Cas9, which can be used to eradicate AMR from microbial communities. However, this technology has only been tested under laboratory conditions, and is not yet ready for use in the real world. My proposed research will take the development of this technology to the next level by testing for the first time whether CRISPR-Cas9 can eradicate AMR from a complex microbial community, isolated from the pig gut. The pig gut microbial community is a highly relevant study system, since decades of consistent overuse of antibiotics in animal industries to boost animal growth and limit disease has selected for very high levels of AMR in the animal gut, causing severe risks for human and animal health. The resulting AMR enters the environment due to the application of animal manure for soil fertilization, which is thought to also contribute to AMR emergence in human populations. In my research I will first develop genetic tools that will help to spread CRISPR-Cas9 through a microbial community. I will monitor the spread of CRISPR-Cas9 and the associated AMR decline. Crucially, I will also carefully monitor the consequences: too often have promising pest and disease control strategies been applied without considering the ecological and evolutionary risks, sometimes leaving a devastating impact on ecosystem functioning and causing resistance to emerge. I foresee two scientific challenges associated with implementing this new technology. First, removal of an AMR gene may cause unwanted or unanticipated increases in other AMR genes that are functionally redundant. Second, the AMR gene that is targeted by CRISPR-Cas9 may evolve to become resistant to targeting, which could undermine the technology. To address these challenges, I will monitorthe changes in a pig gut microbial community caused by the removal of AMR genes, and whether resistance to CRISPR-Cas9 will evolve. This will involve introducing CRISPR-Cas9 to the pig gut community in a long-term (12 months) experiment, and in parallel introducing CRISPR-Cas9 to simpler derived communities in short-term experiments (2 months). For both types of experiments, I will then examine the ecological and evolutionary changes that occur. These experiments will take place in a contained laboratory environment, to avoid unintended release of the CRISPR-Cas9 into the environment. Crucially, the data from these experiments will feed into a mathematical model to generate a theoretical framework that allows me to predict these ecological and evolutionary consequences in other microbial communities. This research will be carried out at the University of Exeter, one of the centers of excellence for studying microbial community ecology, evolution of AMR and CRISPR-Cas9. I will be embedded in a highly collaborative research environment, sharing laboratory space with key experts from the relevant disciplines. The BBSRC Future Leader Fellowship will allow me to start building my own research group, and to become a fully independent researcher. I will use this fellowship as a springboard to attract further funding to expand the scope of my research program, and to pursue novel lines of research that dovetail from this project. AMR is now recognized by the UN General Assembly as one of the most urgent problems that our society is facing, and a key strategic priority for BBSRC research funding. Discovering new strategies to minimize the burden of AMR in both humans and animals would be truly groundbreaking. Testing whether CRISPR-Cas9 can eradicate AMR from a complex microbial community and understanding the consequences of AMR removal will be a major step forward to push such a breakthrough.

Impact Summary

I expect that the scientific outcomes from this research will have economic and societal benefits for a number of stakeholders related to agriculture, industry, academia, and members of the public. Below I will discuss these different stakeholders and how they will benefit from my research outcomes. (1) Agricultural sector The agricultural sector will be the most important stakeholder benefitting from this research. The sector faces massive problems associated with AMR in livestock, mainly as a consequence of the consistent overuse of antibiotics in animal industry to boost animal growth and to limit infectious disease. This has led to the current situation where livestock animals, particularly pigs, carry very high levels of AMR in their guts, which severely exacerbates the risk of the evolution of multi-resistant bacteria. Using the manure of these animals to fertilize soils carries the additional risk of spreading AMR in the environment. This research can inform the sector on the potential of CRISPR-Cas9 plasmids to clear AMR plasmids from a gut community. It also forms the basis for the development of potential plasmid-based mitigation strategies to suppress AMR levels in animal gut microbiota, for example through the use of plasmid-based "probiotics" for livestock (see below). I will disseminate my research through peer-reviewed publications and by giving seminars on national and international conferences. (2) Animal health industry I expect the animal health industry to benefit from this research, as it will give crucial insights in the potential application of CRISPR-Cas9 plasmids to clean animal guts, for example by using these plasmids in the form of a "probiotic" to limit AMR levels in animal guts and to prevent that certain AMR plasmids increase to very high frequencies. (3) Academic stakeholders I expect different academic groups to benefit from my research. In particular, animal scientists and agricultural engineers will benefit as it will help themto improve animal health by reducing the risk of AMR spread in animal guts. Biomedical researchers will be informed on the potential to use this strategy in human guts. Furthermore, this research will inform microbial and biotechnological engineers on the potential of using CRISPR-Cas9 for community engineering. Ecologists working on community stability and ecology will benefit, as will (evolutionary) biologists working on host-symbiont interactions. In addition, microbiologists working on the biology of CRISPR-Cas systems will profit from the advancements of this research. I will disseminate my research to fellow academics through peer-reviewed publications, by giving seminars at national and international conferences (CRISPR conference 2018, 'Environmental Dimensions of Antibiotic Resistance' meeting 2019, ISME meeting 2020, ASM Microbe meeting 2020) and by visiting collaborating universities or institutes (CNRS Montpellier, visit to collaborator Prof. Gandon) (partially funded through this fellowship). (4) The wider public I anticipate that the interest from the general public for my research will be twofold; (i) the general public is starting to realize the gravity of AMR due to intense media coverage (TV documentaries, newspaper articles etc.) in recent years, and (ii) CRISPR-Cas9 is in the public spotlight due to the recent breakthroughs in genome editing. I plan to engage with the public by taking part in the "Science Sessions" on the local radio station where I will explain to the general public the 'what, how and why' of my research. I will also visit one school during my Fellowship to enthuse children about science in general and research on AMR in particular. I will schedule this visit to coincide with the WHO Antibiotic Resistance Awareness Week, which usually takes place each year in November.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	Fellowship - Future Leader Fellowship (FLF) [2014-2015]
Funding Scheme	X – not Funded via a specific Funding Scheme

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