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

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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.