The CRISPR system: a new frontier in prokaryotic molecular biology

The battle between viruses and the cells they attempt to infect and subvert is one of the main driving forces in molecular evolution. Prokaryotes target and degrade invading DNA using 'restriction enzymes', the discovery of which revolutionised molecular biology. Eukaryotes, including humans, use the RNA interference (RNAi) pathway to target and degrade viral RNA. In the last few years a prokaryotic version of the RNAi pathway, the so-called CAS-CRISPR system, has come to light. Viral sequences are stored in the host genome as 'spacers' flanked by direct repeat sequences (CRISPRs). It is known that host cells incorporate new viral DNA into the CRISPRs, and that this confers immunity against future viral infection. The CRISPR system is envisaged to act in an analogous manner to the RNAi pathway A number of widely conserved 'Cas' proteins (up to 50 per genome) are associated with the CRISPR sequences, and these are thought to be responsible for CRISPR processing, viral defence and the incorporation of new viral sequences. The functions of these proteins are not clearly understood. We have the experience and technological know-how to study all the Cas proteins from the model organism Sulfolobus solfataricus. Cas protein structures will be solved, and Cas protein interactions delineated. The activities of individual Cas proteins and complexes will be determined using a variety of techniques in routine use in our laboratories. The ultimate aim is to acheive a molecular understanding of the CRISPR system. As well as providing fundamental new insights into prokaryotic biology, the work may lead to new methods to manipulate the genomes of useful organisms (synthetic biology) or to treatments for important microbial pathogens.

The Cas-CRISPR system is a widely conserved prokaryotic phage defense system that has only recently been recognised as such. Many bacterial and archaeal species store short sequences of phage DNA as spacers in tandem arrays with conserved repeat sequences (CRISPRs). These confer immunity against infection by phage with the cognate sequences. The mechanism is thought to be analogous to RNA interference in eukaryotes, but specific details of the pathway are not yet known. The CRISPR arrays are associated with Cas proteins that have been predicted as nucleases, helicases, polymerases and RNA binding proteins. Together the Cas proteins are thought to process CRISPRs to generate prokaryotic short interfering RNAs (psiRNAs) that mediate sequence specific degradation of phage mRNA's. There must also be a mechanism to incorporate new phage sequences into the CRISPR arrays, but the mechanism is completely unknown. The primary aim of this work is to delineate the functions of the different Cas proteins in the defense against invading phage, using the model organism Sulfolobus solfataricus, which has over 50 Cas proteins. All proteins will be cloned and subjected to crystallisation trials and structural studies. Protein interactions will be analysed and the activities of individual proteins delineated. The prokaryotic equivalents of the Slicer and Dicer proteins from the RNAi pathway will be identified and characterised. The function of the large multiprotein Polymerase Cassette Complex (PCC), which we have recently purified, will also be tested.