Cyclic oligoadenylate signalling - a new type of antiviral response

All living things have evolved the ability to respond to changes in their environment in a way that maximises their fitness. For example, bacteria swim towards a food source, and away from a harmful chemical. To achieve this, they need a way to convert an environmental signal into a signal inside the cell, and they do this with a class of molecules called "second messengers". In 2017 an entirely new class of second messengers was discovered in bacteria and archaea: cyclic oligoadenylates (cOA). cOA molecules are made by joining together molecules of Adenosine triphosphate (ATP) to form rings of 3, 4, 5 and 6 building blocks. The enzyme that makes cOA is a cyclase that is part of a large complex important in the CRISPR system. This effector complex, which goes by several names (Csm, Cmr, Type III) can sense the presence of a virus in the cell by binding specifically to its genetic material. When the viral RNA is bound, the cyclase is switched on and cOA second messengers are synthesised to signal to the cell that it is infected. This sets in chain an antiviral response that includes changes in gene expression and activation of ribonucleases (RNA cutting enzymes) that degrade RNA in the cell. This might help buy some time for the cell to kill the virus, or alternatively might push the cell into dormancy or even death. Any of these outcomes, while not necessarily good for the cell, can be good for the cell's neighbours. And as these neighbours tend to be related, this process is favoured by evolution as it stops infection spreading.

In this grant, we propose to study this new cOA signalling system in a model organism known as Sulfolobus solfataricus - which is found in volcanic pools, thriving at high temperatures and acidic conditions. Sulfolobus is an ideal model system for the biochemical, genetic and structural studies we want to carry out. It has a well understood CRISPR system and a good number of proteins activated by the cOA signalling molecule. We will examine how cOA is synthesised and degraded, and how it binds to and activates the downstream proteins that elicit the antiviral response. The work will help us to understand the fundamental properties of this exciting new antiviral signalling system. Since many crop and human pathogens have CRISPR type III systems, this work could ultimately find application in new methods to combat these diseases. The proposal fits well with the BBSRC strategic priority area: Integrative Microbiome Research.

In 2017, two groups studying the CRISPR system for antiviral defence discovered an entirely new class of signalling molecule: cyclic oligoadenylate (cOA). cOA is synthesised by joining 3-6 molecules of ATP into a ring structure - an activity catalysed by the cyclase domain of the Cas10 protein. Cas10 is part of the type III effector systems (also known as Csm and Cmr), which are programmed by binding a CRISPR RNA (crRNA) molecule to allow them to detect invading genetic entities. Once viral RNA is detected, the type III systems activate RNA cleavage, DNA cleavage and the synthesis of cOA. cOA in turn activates one or more downstream proteins with a cOA-binding (CARF) domain. Some of these CARF domain proteins are transcription factors, which sculpt the gene expression profile in response to viral infection. Others are nucleases, which, once activated, degrade RNA. It is not yet clear whether viral RNA is targeted specifically, or whether degradation is non-specific, leading to a general shut-down of gene expression, dormancy and potentially death - all responses that might be appropriate for an infected cell. Here we propose to study the cOA signalling system of the model organism Sulfolobus solfataricus. We have shown that S. solfataricus can make cOA, and have developed a way to make defined cOA analogues easily. We have demonstrated that cOA activates a ribonuclease in the cell, and identified the transcription factors that are activated. We will dissect the cOA signalling system with a view to understanding the synthesis and degradation of cOA molecules, the way they activate CARF domain proteins, and the consequences for gene expression and RNA turnover. These findings will have general significance of the wide variety of bacterial and archaeal species with a type III CRISPR system and a cOA signalling pathway. This includes many human and crop pathogens, where new avenues for control may ultimately become possible.

Public Engagement
The PI is committed to activities supporting the public understanding of science - evidenced by a track record of engagement with Schools, public lectures and science festivals such as the Cheltenham science festival and Association for Science Education. School pupils will also get an opportunity to attend the laboratory and gain work experience (average of 1 student per year in the last 3 years). We will undertake a series of visits to secondary schools, comprising a talk on CRISPR followed by a debate held by the pupils on the ethics of various potential applications - such as germline manipulation. We will develop briefing materials suitable for schools to help illustrate these issues, consulting with science teachers on how this material can fit into the national curriculum. This theme will also be delivered at science fairs by development of an interactive quiz that highlights the relevant issues in an engaging way.
Research and professional skills
The PDRA employed on the grant will receive extensive training in a variety of disciplines spanning microbiology through biochemistry to biophysics. They will have access to the award winning courses run by the University of St Andrews, which aim to provide a wide variety of transferrable skills. By the end of the project they will have acquired the "Passport to Research Futures" a structured development programme tailored by St Andrews revolving around career planning, professional development and employability.
Economic and Societal Impact
Fundamental scientific research on the CRISPR-Cas system has had a highly significant (and at the time of funding decisions largely unforeseen) impact on broad area of the economy, including biotechnology and developments in health care. There has also been a strong societal impact, for example through the ethical challenges that arise when the technology for germ line gene editing becomes facile. We cannot predict where the work proposed in this application will take us in terms of impact, but we are open to all possibilities. Type III CRISPR-Cas systems, though more complex than Cas9, are very powerful gene editing machines in their own right. The cOA signalling pathway could potentially be subverted for the control of animal and crop pathogens. We will work with our dedicated Business Development Manager to explore all opportunities for the maximisation of applied outcomes arising from this research.