Elucidating the molecular architecture of the Archaeal CMR complex, a key player in the unicellular immune response.

We want to understand the fine detail of a newly discovered mechanism that unicellular organisms belonging to Bacteria and Archaea have developed as a defense from viral attack. This inheritable mechanism, termed CRISPR system, is present in a number of organisms, some of which cause disease in plants and animals. As an example, a well-characterized CRISPR belongs to the bacterium which is the main cause for dental cavities.

The function of proteins is directly linked to their three-dimensional structure. We will determine and interpret the structure of a key molecular machine belonging to the CRISPR system. This multi-component assembly orchestrates the silencing of viral RNA in the Archaeon Sulfolobus solfataricus, a model organism in the study of CRISPR. To do so, we will use electron microscopy coupled to image processing. This technique allows to acquire projection images of isolated assemblies, and to use them to calculate their 3D shape, therefore providing important insight on the molecular mechanisms used as defense from viral attack.

This study is biologically important because it is the key to understand how a large number of living organisms counteract viral infection. In the longer term, it also has a strong potential in biotechnology, since it could be exploited to specifically target disease-related microorganisms.

This research will be performed at the School of Biological Sciences in Edinburgh, where state-of-the-art resources for structural electron microscopy have been recently established, in collaboration with the Biomedical Sciences department in St Andrews, a centre of excellence in the molecular biology of Archaea.

We propose to perform structural electron microscopy studies of a recently discovered mechanism, which Archea and Bacteria use against viruses and phages.

CRISPRs ("Clustered Regularly Interspaced Short Palindrome Repeats") are a distinct class of repetitive elements, which are present in over 40% of prokaryote genomes, and consist of a direct repeat of 28-40 bp separated by 25-40 bp spacers. These DNA repeats do not encode proteins, but they are transcribed to generate long RNA precursors that are processed into monomers and/or multimers of the repeat motif. The so-called Cas genes (for CRISPR-associated) are a complex array of protein-encoding genes associated with CRISPRs. They encode proteins for DNA/RNA processing, such as helicases and nucleases. The Cas machinery is classified into a group of core Cas components (Cas1-6) and a set of CMR module proteins (Cmr1-7). CRISPR/Cas and Cmr modules are usually located in the most variable regions of chromosomes and are often displaced as a result of genome shuffling.

CRISPR and Cas/Cmr induce immunity with concerted adaptation, expression and interference mechanisms. To gain mechanistic insights into this important defense against exogenous genetic material, we want to elucidate the CRISPR/CMR machinery involved in RNA interference in Sulfolobus solfataricus with an approach based on electron microscopy and single particle analysis.

This research will be a concerted effort between the Spagnolo laboratory in Edinburgh and the White laboratory in St Andrews.

Academic impact
- This basic research proposal on the CRISPR system will have an impact on scientists working on host-pathogen interactions as well as on the molecular biology of Archaea, where the White group have an internationally recognized leading role.
- The structural electron microscopy initiative at the University of Edinburgh is very recent, and therefore it bears a strong component of staff training. LS will contribute to furthering the training of the named RA on the grant, as well as of future PhD and project students. This is crucial to maintaining the fundamental role in structural biology traditionally held by the UK.
- The number of female PIs in Science, Engineering and Technology (SET) has been historically quite low. As a female PI in structural biology, LS hopes she will act as a mentor and role model to students, and in particular female students who are keen to develop an academic career in SET.

Outreach
- The Edinburgh group plan to organize a workshop aimed at final year school pupils, with the ultimate goal of engaging them both with the biological question and with the research methods used. Funds have been requested for this.
- The St Andrews group have already engaged in several outreach initiatives, which they plan to continue with annual lectures in Schools and public understanding of science events.

Research and professional skills
- Obtaining support for this grant will allow two very valuable members of staff to be retained. Additionally, the named RA on this grant will have an opportunity to further develop her skills in structural electron microscopy, therefore extending her portfolio of expertise. She will be involved in people management, through the supervision of project students. The University of Edinburgh offers a wide range of training on portable skills, including the "Researcher Development" and the "Research Leader" programmes. Among the specific modules, there are courses on Career management, Business and management skills, Communication and dissemination.

Economic and Societal Impact
- Prokaryotic gene silencing. If the CMR complex can be targetted to cleave specific cellular RNAs in prokaryotes, this would allow the development of a prokaryotic RNA silencing technology that would be entirely novel. Consideration of the impact of the equivalent technology in eukaryotes underlines the potential significance of this possibility. The structural studies proposed in this grant will vastly improve our understanding of the mechanism and essential components of CMR and thus bring this possibility closer.
- Phage therapy, the application of bacteriophages to combat bacterial infections, has recently regained interest due to the increase of bacterial resistance to conventional antibiotics. A thorough understanding of the CRISPR system could lead to advances in the engineering of anti-bacterial phages.
- The subject of CRISPRs, which relates to the "arms race" between viruses and cells and also to unusual Lamarckian modes of evolution, are particularly interesting for lay audiences.