CRISPR-mediated DNA cleavage by the CSM complex

The CRISPR system is a recently discovered antiviral defence system present in many microbes. CRISPR proteins use small RNA's to detect and destroy invading viruses with similar sequences. CRISPR functions as a adaptive immune system in microbes, analogous to the human immune system. The machinery used to seek out and destroy invaders can be classified into three major types, I, II and III. CRISPR type II has been used very successfully to target specific genes for genome engineering applications in many different organisms and shows great biotechnological and medical potential. The type III system is much less well understood but could provide an alternative technology to pursue the same applied aims. We have recently published the first detailed description of a CSM (type IIIA) CRISPR complex and we now wish to pursue a detailed understanding of the mechanism by which this complex binds RNA and target DNA species, how degradation of target DNA happens and to understand how the structure of the complex relates to its functions. The findings will be of fundamental scientific interest and may also allow the development of new technologies for specific gene targeting.

CRISPR (clustered regularly interspaced palindromic repeats) is a recently discovered prokaryotic antiviral defence system. CRISPR loci in the genome store a record of past viral infection and act as an immune system. Transcription of these loci yields CRISPR RNA (crRNA) that is used by large, multisubunit ribonucleoprotein complexes to detect and degrade invading genetic elements. These CRISPR associated (Cas) complexes are typically up to 400 kDa in size and their structures and mechanisms are not fully understood. We have been funded by BBSRC since 2009 to study the CRISPR system, one of the very few labs in the UK in this area. In the current application we focus on the CSM (Type IIIA) Cas complex, which is the least well-understood CRISPR pathway. We recently published a detailed description of the subunit organisation and structure of the complex (Zhang, Mol Cell 2013). We now wish to work towards a molecular understanding of the structure and catalytic mechanism of the complex. Specific aims are to gain an understanding of crRNA binding by the complex, to define the dsDNA binding and cleavage mechanisms and to glean a greater understanding of the structure of the complex and its constituent parts.The CSM system, though more complex than Cas9, could provide an alternative method for targeted gene disruption. Until the fundamental science proposed here is carried out, we cannot determine whether there are practical advantages such as lower off-target effects for CSM over Cas9. This application seeks to lay the fundamental scientific groundwork that will help determine the structure, mechanism and potential applications of the CSM system.

Academic impact
Scientists working in academia and industry will benefit from the basic advances in our understanding of the mechanism of CSM-mediated specific DNA cleavage. There is the potential for new tools allowing targeted gene disruption in cells. The UK scientific community does not have a strong presence in the CRISPR field.
Outreach
The public, particularly young people, will benefit from outreach activities linked to this application. The PI is committed to activities supporting the public understanding of science - evidenced by a track record of engagement with Schools, public lectures and science festivals such as the Cheltenham science festival. School pupils will also get an opportunity to attend the laboratory and gain work experience (average of 1 student per year in the last 3 years). The team employed on the grant undertake to prepare and deliver an exhibit for science festivals (funds are requested for this). The subject - the battle between viruses and cells and its role in evolution - lends itself to public engagement opportunities. This work will expose the public, particularly young people, to exciting science and enhance their understanding of evolution.
Research and professional skills
The project will support two key members of staff in St Andrews. Dr Shirley Graham is pursuing a career as a research technician and is currently BBSRC-funded. This grant will secure her funding for a further 28 months. The unnamed PDRA will likely be a recently qualified PhD student with some relevant background skills. The PDRA will receive extensive training in a variety of disciplines spanning microbiology through biochemistry to biophysics. Dr Huanting Liu is a senior researcher and will be funded 30% on the grant. Publications arising from this project will help him to develop his career as an independent scientist. All three researchers will have access to the award winning "Gradskills" courses run by the University of St Andrews, which aim to provide a wide variety of life skills. The grant, if funded, will also underpin the research of a PhD student, BBSRC funded, working on the CRISPR system in the White laboratory.
Economic and Societal Impact
In the medium to long term, Industry, including biotech and fermentation companies, stand to benefit from the knowledge and technology that will result from this project. CRISPR technology has made great progress in recent years and there is a danger that the UK is being left behind in this area - very few research groups are funded to study the system in the UK.