Dissecting the Molecular Biology of Cyclic Oligoadenylate Signalling

Just like humans, bacteria have a complex immune system to protect them against infection by viruses. The CRISPR system provides adaptive immunity in bacteria, analogous to our antibody system. For the public, CRISPR is often synonymous with the Cas9 enzyme that has allowed rapid advances in genome editing technology. However, Cas9 (a type II CRISPR system) is quite rare in microbes, which more frequently have type I or type III CRISPR systems. Recently, scientists studying type III CRISPR discovered a novel signalling molecule, cyclic oligoadenylate (cOA), that is built from the main energy molecule in the cell, ATP. cOA signals an infected state in cells exposed to a virus, mobilising the cell's defences. We only know about one aspect of this defence - an enzyme that chews up RNA, called Csm6. It seems to buy the cell some time to help clear the infection, but may also cause the cell to "commit suicide" if it can't get rid of the virus. By studying the CRISPR system in different bacteria, we have discovered two new cellular defence enzymes that are activated by the cOA signal to degrade viral genetic material. We have also identified an enzyme made by viruses that rapidly breaks down cOA - it is probably acting as an "anti-CRISPR" to shut down the cell's defences. We want to understand how these three enzymes bind to cOA, how they assume an active shape and what they do, both in vitro (in the test-tube) and in vivo (in the cell). This work will provide a step change in our understanding of an important cell defence pathway. There are also potential applications in biotechnology and human health, in particular for new approaches to target drug resistant bacteria using viruses.

The CRISPR system provides adaptive immunity for prokaryotes against mobile genetic elements (MGE). Recently, type III CRISPR defence systems were shown to generate 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 when foreign RNA is detected in the cell. cOA is a potent signalling molecule that binds downstream effectors via a CRISPR-associated Rossman Fold (CARF) domain. The only characterised example is the degradative ribonuclease Csx1/Csm6 which destroys both invader and host RNA. However, many organisms encode further/ alternative predicted CARF-domain effector proteins with unknown function. Here we build on significant preliminary work to dissect the structure, activation, mechanism and specificity of two novel CARF-family effector proteins: Tthb155 and DUF1887. The signalling molecule cOA must be removed to deactivate the CARF effector protein defences. We previously showed how Sulfolobus does this by slow turnover using a "Ring Nuclease". We have recently identified a novel and widely distributed viral-encoded anti-CRISPR protein which we term the "viral Ring Nuclease" (vRN). vRNs bind cOA using a fold distinct from the CARF domain, and turnover the signalling molecule very quickly, potentially deactivating the cell's defences. We aim to understand the molecular mechanism and specificity of this enzyme family and explore how it provides immunity against the cellular defence system. We have developed a modular recombinant type III CRISPR system in E. coli to facilitate in vivo studies of these novel enzymes, and have initiated key new collaborations with industry and academia to explore the development and uses of cyclic nucleotide analogues. The work has potential application in the development of new diagnostic technologies and new approaches to the treatment of antibiotic resistant bacteria.

Academic impact:
The multi-disciplinary nature of the proposed research means that it should be of interest to academics from a range of backgrounds, including microbiologists, molecular biologists, biochemists and structural biologists, both in academia and industry. There is a strong CRISPR community within the UK, as well as internationally. The project will facilitate new international collaborations with both academia (Prof Yitzhak Tor, UC San Diego) and industry (Dr Frank Schwede of BIOLOG Life Science Institute Bremen) that will maximise the impact of the science carried out. The research will also contribute to the research and professional training of the PDRA and Dr Verena Oehler (research technician). They, and the PIs, will have access to the University's suite of courses and workshops for development of life skills and for research. Staff working on the proposed project will obtain diverse laboratory skills, which can be translated to a host of other biological questions and therefore important for future employment opportunities. In addition, staff will learn skills, such as oral presentations and time planning, which can be translated to other employment sectors. Dr Verena Oehler is currently employed at the University of St Andrews on a fixed term contract, meaning a successful outcome for the grant would enable the retention of talented and experienced staff at the University of St Andrews.

Economic impact:
Fundamental scientific research on the CRISPR system has already had a highly significant impact on broad areas of the economy, including biotechnology and developments in health care, and this will continue given the amount still to understand about these systems. Type III CRISPR systems, though more complex than Cas9, are very powerful gene editing machines in their own right. Aspects of the cOA signalling pathway have recently been exploited in the development of new diagnostic tools such as SHERLOCK, which utilises the Csm6 ribonuclease. The new cOA dependent enzymes we are characterising have potential applications in this arena. A further strong area of applied activity is in the development of next generation phage therapy approaches to tackle the scourge of multi-drug resistant bacteria. A patent application has been filed covering uses of the vRN protein family in this area and we will work with the Knowledge Transfer Centre at the University of St Andrews throughout the grant period to explore all opportunities for the maximisation of applied outcomes.

Societal impact:
CRISPR technology is already having a strong societal impact, for example through the ethical challenges that arise as the technology for germ line gene editing becomes facile. We aim to contribute societal impact through education of the public sector, in particular children and their families, by the development of a new activity focused on the underlying principles of the CRISPR system and its potential exploitation in gene editing. The activity will encompass visual displays, models to help explain the concepts and use augmented reality technology to visualize relevant protein structures. This activity will be delivered at science festivals, local schools, in particular those in underprivileges regions of Fife, and at First Chances events at the University. Both PIs have a strong track record in the delivery of public engagement activities.