CRISPR-Cas9 gene drives to fight antimicrobial resistance
Lead Research Organisation:
UNIVERSITY OF EXETER
Department Name: Biosciences
Abstract
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 monitor the 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.
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 monitor the 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.
Technical Summary
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.
Planned Impact
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 them to 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.
(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 them to 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.
Publications
Chabas H
(2018)
Evolutionary emergence of infectious diseases in heterogeneous host populations.
in PLoS biology
Chevallereau A
(2019)
The effect of bacterial mutation rate on the evolution of CRISPR-Cas adaptive immunity.
in Philosophical transactions of the Royal Society of London. Series B, Biological sciences
Chevallereau A
(2020)
Exploitation of the Cooperative Behaviors of Anti-CRISPR Phages.
in Cell host & microbe
Common J
(2019)
CRISPR-Cas immunity leads to a coevolutionary arms race between Streptococcus thermophilus and lytic phage.
in Philosophical transactions of the Royal Society of London. Series B, Biological sciences
Common J
(2020)
Diversity in CRISPR-based immunity protects susceptible genotypes by restricting phage spread and evolution.
in Journal of evolutionary biology
| Description | 1. During my fellowship I developed pKJK5::Cas, a broad host-range plasmid which encodes cas9 and an sgRNA to protect its hosts from AMR plasmid uptake. Applied conjugatively, my work demonstrated that pKJK5::Cas was able to remove resident AMR plasmids from recipient bacteria - the extent of this removal was dependent on conjugation efficiency and CRISPR targeting efficiency. 2. Additionally, the presence of toxin-antitoxin addiction systems on target plasmids and target plasmid incompatibility protected from their removal. 3. When allowed to spread in a synthetic bacterial community, pKJK5::Cas became lost in a community-dependent manner, due to its fitness cost being influenced by the presence of certain bacterial community members. 4. Large-scale bioinformatics analyses show that CRISPR-Cas systems negatively associate with AMR genes in a range of human pathogen species. However, there was also significant variation in the strength of this association across species, which could partially be contributed to the specific associations of different CRISPR-Cas types with aMR genes. Overall, my work shows that CRISPR-Cas9 removal of AMR plasmids in microbial communities is achievable when either conjugation efficiencies are high enough, or fitness costs are very low, and both of these are dependent on microbial community context. Imposing positive selection on the CRISPR-Cas9 plasmid will likely be very important to increase the spread of the CRISPR-Cas9 plasmid, and thereby its AMR removal efficacy, which will the subject of future investigations. |
| Exploitation Route | 1. My work shows imposing positive selection on the CRISPR-Cas9 plasmid will likely be very important to increase the spread of the CRISPR-Cas9 plasmid, and thereby its AMR removal efficacy. 2. The finding that the presence of a microbial community strongly influences the cost of plasmid carriage and its long-term maintenance in a focal species is an important finding, as it shows that interspecific competition can have a destabilising effect on plasmid maintenance; knowledge which may be leveraged in clinical and natural environments to cure plasmids from focal species. |
| Sectors | Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Discussed with scientific advisors about the opportunities and challenges around the use of phage theray and CRISPR-Cas based tools in AMR. |
| Geographic Reach | Europe |
| Policy Influence Type | Participation in a guidance/advisory committee |
| Description | Lister Prize Fellowship |
| Amount | £250,000 (GBP) |
| Organisation | Lister Institute of Preventive Medicine |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 09/2021 |
| End | 08/2027 |
| Description | Microbiota Intervention Strategies Limiting Selection and Transmission of Antibiotic Resistance burden in the One Health domain |
| Amount | £505,351 (GBP) |
| Funding ID | MR/W031191/1 |
| Organisation | Medical Research Council (MRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 02/2022 |
| End | 01/2025 |
| Title | Diversity in CRISPR-based immunity protects susceptible genotypes by restricting phage spread and evolution |
| Description | Diversity in host resistance often associates with reduced pathogen spread. This may result from ecological and evolutionary processes, likely with feedback between them. Theory and experiments on bacteria-phage interactions have shown that genetic diversity of the bacterial adaptive immune system can limit phage evolution to overcome resistance. Using the CRISPR-Cas bacterial immune system and lytic phage, we engineered a host-pathogen system where each bacterial host genotype could be infected by only one phage genotype. With this model system, we explored how CRISPR diversity impacts the spread of phage when they can overcome a resistance allele, how immune diversity affects the evolution of the phage to increase its host range, and if there was feedback between these processes. We show that increasing CRISPR diversity benefits susceptible bacteria via a dilution effect, which limits the spread of the phage. We suggest that this ecological effect impacts the evolution of novel phage genotypes, which then feeds back into phage population dynamics. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| URL | http://datadryad.org/stash/dataset/doi:10.5061/dryad.66t1g1k00 |
| Description | Interviews for New Scientist, Science Daily, The Atlantic |
| Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Media (as a channel to the public) |
| Results and Impact | I've done several interviews on our paper on cooperation between anti-CRISPR phages that was published in 2018. These include the New Scientist and the Atlantic, and 10 other online news channels. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Pint of Science |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | Gave a lay talk about our work on CRISPR-Cas and antibiotic resistance during a Pint of Science event in a local pub in Falmouth. |
| Year(s) Of Engagement Activity | 2019 |
| Description | stand at Royal Cornwall Show on antibiotic resistance |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | We set up a stand at the Royal Cornwall Show to explain the audience (mainly kids with their parents) about what antibiotic resistance is, how bacteria become antibiotic resistance, what individuals can do about it, and how our research contributes to solving the problem. |
| Year(s) Of Engagement Activity | 2019 |