‘CARFing’ out the signalling network of CRISPR-Cas

CRISPR-Cas technology has had a tremendous impact on the genome editing field, stimulating fundamental research and resulting in applications, ranging from biotech to human gene therapies. Similar to many other genetic tools, this technology originates from prokaryotes, where it functions as an adaptive immune system to combat infection by viruses. Upon such an infection, CRISPR-associated (Cas) proteins use guide RNAs to specifically recognize and cleave invading nucleic acids. The programmable nature of the guide RNAs has allowed for repurposing the Cas proteins into specific DNA (or RNA) cleaving enzymes. However, apart from the well-known Cas9, there is a huge diversity of CRISPR-Cas systems. The underlying unique features of some of these other systems and their biological significance remain to be discovered. A good example is the recent discovery of a signal transduction network in type III CRISPR-Cas systems, involving the production of a second messenger molecule that activate proteins in this network (called CARF proteins). Bioinformatic analyses predicted that the list of CARF proteins and the different catalytic activities associated with them is abundant and highly diverse.

In this project, we aim to develop a thorough understanding of CARF proteins in the type III signalling network and repurpose some of them for unique applications. Firstly, we will characterize how CARF proteins are activated and how they contribute to providing a robust response against virus predation using the model bacterium Thermus thermophilus. Secondly, we will biochemically and structurally explore the wealth of different catalytic activities of CARF proteins that exist in nature. Lastly, we will repurpose a subset of these for innovative molecular diagnostics and base editing platforms. The outcome of this project will therefore be of high scientific interest (understanding the biology behind the CRISPR-Cas signalling network) and will spark a new range of applications for medical and applied sciences.