Project
‘CARFing’ out the signalling network of CRISPR-Cas
CRISPR are prokaryotic immune systems associated with nucleic acid degradation. Here, we explore a newly discovered CRISPR-associated signalling system, its multitude of enzymatic activities, and their repurposing for genome engineering.
Background
Among the six known CRISPR-Cas system types, type III systems are arguably the most complex and versatile to date. Aside from being capable of degrading both RNA and DNA targets, these systems produce signaling molecules upon target detection that tightly control a range of so-called CARF and SAVED proteins, with novel activities believed to be involved in defense against mobile genetic elements. Some of these proteins indiscriminately cleave both invading and host nucleic acids, while others exhibit activities not yet seen in CRISPR-Cas defense, such as NAD+ degradation and protein cleavage. Some of these activities are now being exploited to develop novel tools that require tight control upon target detection, such as in diagnostics. Interestingly, there remains a plethora of uncharacterized CARF and SAVED proteins with a diverse range of domain architectures predicted to be activated by type III systems using signaling molecules.
Aim of the project
This project will have both a fundamental and applied aspects. For the fundamental aspect, we aim to characterize predicted CARF and SAVED proteins, their enzymatic activities, and how these contribute to immune defense. Following this, we plan to repurpose these proteins for different applications, such as in diagnostics or gene editing, taking advantage of the tight control of type III CRISPR-Cas systems over their activities.
Techniques
For this project, we will work mostly with E. coli for strain engineering and protein production. Depending on the project, you can gain expertise on the following techniques: molecular cloning, protein purification, in vitro and in vivo growth and interference assays.
Contact
Are you an enthusiastic BSc or MSc student who wants to learn more about and work on this novel signalling system underlying CRISPR-Cas systems? Feel free to contact me via carl.salazar@wur.nl.
