Mechanisms and evolutionary consequences of host immunosuppression by anti-CRISPR phages

CRISPR-Cas is an immune system present in many bacteria that protects them against infections with bacterial viruses, called phages. Several years ago researchers made the exciting discovery that phages have evolved ways to counteract this immune response, by producing small proteins that bind to CRISPR-Cas immunity complexes and thereby block their activity. These molecules, named anti-CRISPRs (Acr), are produced immediately upon phage infection, but our earlier work has shown that Acr production is often 'too little, too late" so that phages are outpaced by the CRISPR-Cas complexes in the bacterial host cell. However, our work also shows that Acr linger on in the cell and keeps blocking CRISPR-Cas immunity complexes, even when the initial phage infection has been cleared. This opens the door to a second phage that can now successfully infect this immunosuppressed host. Some Acrs are "stronger" than others, i.e. they are better at blocking CRISPR-Cas, but we do not understand what factors determine how strong an Acr is and the associated immunosuppression. Being able to strongly immunosuppress is not necessarily better for the phage; when different phage species compete with each other (which is very common in many environments), phages that do not produce Acr can exploit those that produce strong Acr molecules. Our data indicate that Acr are costly to produce (for example, because it takes energy to produce them) and this exploitation risk could therefore mean that during phage-phage competition the fitness of the phage producing Acrs is lowered. In the proposed project we wish to understand the mechanism by which Acr-phages immunosuppress their hosts, and the evolutionary consequences of host immunosuppression on maintenance of Acr genes in the absence and presence of phage-phage competition. We will do this by integrating research on the molecular level (how do Acr bind to CRISPR-Cas immunity complexes?), the single-cell level (how does the ability to immunosuppress influence the infection success of phage in individual bacteria? How does this change when phages that infect the same bacterium compete with each other?) and the population level (are Acr molecules costly? is it always better for a phage to produce multiple different Acrs over one Acr? and how does phage-phage competition influence phage fitness and therefore Acr gene maintenance in the long term?). The research outcomes are important for several reasons. Understanding how Acrs interact with CRISPR-Cas complexes and how natural selection acts on them is important for our fundamental understanding of CRISPR-Cas biology. CRISPR-Cas also has extremely important applications, as they are extremely useful tools in a technique called gene editing, where specific mutations or genes can be removed or introduced in the DNA of an organism. This has major potential in healthcare, where the technique could be used to repair mutations that cause genetic diseases in human, such as Cystic Fibrosis, Duchenne muscular dystrophy and Huntington's disease. But CRISPR-Cas is also used for ecological engineering, for example to reduce the spread of infectious diseases by insects, and for microbiome engineering to remove antimicrobial resistance and/or pathogenic bacteria. For these applications, it would very useful to have ways to control CRISPR-Cas activity (for example, because we only want it to be active during a certain time period or in specific tissues or cells). Currently, we have no ways to control CRISPR-Cas activity on protein level, but using Acr molecules would be an extremely reliable way of temporal inactivation of CRISPR-Cas as required. This research will give us new, exciting and important insights in the mechanisms underlying Acr - CRISPR-Cas interactions and the evolutionary consequences of these interactions, which will be crucial for successfully applying Acrs as a CRISPR-Cas regulating tool in healthcare, life sciences research and agriculture.

The discovery of anti-CRISPRs (Acrs), small proteins encoded by bacteriophages that block CRISPR-Cas immunity, has been one of the most exciting discoveries in CRISPR-Cas research of the last 5 years. Understanding how Acrs interact with CRISPR-Cas complexes and how natural selection acts on them is not only important for fundamental research, but Acrs are also extremely promising in situ modulators of CRISPR-Cas activity in genome editing, gene transcription regulation, gene drives and more recently, microbiome engineering. Currently, CRISPR-Cas activity can only be regulated on RNA level, but regulation of its activity on protein level would be an extremely reliable way of temporal inactivation of CRISPR-Cas as required. Even though major steps forward have been made to unravel the mechanisms by which Acrs block CRISPR-Cas immunity, we lack a basic understanding of when Acrs are important and what their consequences are for the CRISPR-Cas systems they antagonize. My earlier work has demonstrated that bacteria can rapidly drive phage extinct by evolving CRISPR-mediated phage resistance, but phage extinction can be avoided by equipping phage with acr genes. However, my recent work reveals that Acrs are imperfect and show a high degree of variation in their strength, i.e. their ability to block CRISPR-Cas immunity. If we are to use Acrs as CRISPR-Cas modulating tool, we need to understand what defines the success of an Acr-phage. Here I will examine the molecular and physiological determinants of variation in Acr strength and how this relates to natural selection acting on Acr-phages, using a combination of biochemical assays, single-cell microscopy and fitness assays. This research will give us unprecendented insights in the causes and consequences of the immense variation that we see between Acrs, and this information will be crucial for successfully applying Acrs as a CRISPR-Cas regulating tool in healthcare, life sciences research and agriculture.

A number of stakeholders related to academia, agriculture, industry, and members of the public are identified that will benefit from the research outcomes.
(1) Food sector
Food-borne pathogens cause a large burden to the healthcare system. Various industries are now starting to use CRISPR-Cas to tackle food-borne pathogens, either by direct targeting (causing pathogen death) or by removing antimicrobial resistance genes from food-borne pathogens in order to resensitize them to antibiotics. The unwanted interference in this process by anti-CRISPRs is a major concern for the successful implementation of this technology. The proposed research helps to develop this technology by increasing our understanding of how Acrs successfully limit CRISPR-Cas activity. Industry has already shown interest in this proposed research as indicated by the attached LoS from Folium Science, a start-up company based in the UK that aims at developing CRISPR-Cas technology to remove pathogens from the food chain. Research outcomes will be disseminated through peer-reviewed publications and by giving seminars on national and international conferences.
(2) Animal industry
The animal industry faces massive problems associated with antimicrobial resistance (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. CRISPR-Cas has been proposed as a new technology to clear AMR plasmids in a gut community, and this research can further help to develop this technology by increasing our understanding of how Acrs can limit CRISPR-Cas activity. Such knowledge is important for two reasons: 1) Acrs could be used as part of a CRISPR-Cas technology to (temporarily and/or spatially) block CRISPR-Cas cleavage activity; 2) Acrs encoded in a complex microbial community might cause unexpected and/or unwanted limitations to the success of these CRISPR-Cas based technologies. Research will be disseminated through peer-reviewed publications and conference seminars.
(3) Academic stakeholders
Academics working in a wide range of fields will benefit from this research. Firstly, CRISPR-Cas fundamental science will benefit as this work will greatly deepen and expand our knowledge on the cellular factors influencing Acr/CRISPR-Cas interactions and the infection dynamics associated with Acr-encoding phages. Furthermore, this research will inform microbial and biotechnological engineers on the strengths and limitations of Acrs as natural suppressors of CRISPR-Cas activity for use in community engineering. Given the importance of CRISPR-Cas as tool in genome editing, my research will also benefit academics who aim to put limitations to CRISPR-Cas based gene editing and/or ecological engineering, e.g. in those instances where genome editing is only wanted under certain spatial-temporal conditions. Research outcomes will be disseminated to fellow academics through peer-reviewed publications and seminars at (inter)national conferences. In addition, the PI is co-organizing a scientific meeting on CRISPR-Cas ecology and evolution in 2019, and a wide range of academics working on various aspects of CRISPR-Cas research will participate.
(4) The wider public
Due to the breakthroughs of the past years in genome editing and gene drives CRISPR-Cas is continuously in the public spotlight. The wider public is a key stakeholder of the proposed research and outcomes will be disseminated by taking part in the "Invisible Worlds" outreach program where we explain the importance of microbiology research to children, and writing a blog for the Infectious Diseases Hub.