CRISPR Adaptation - the basis for prokaryotic adaptive immunity

The CRISPR system is an adaptive immune system in microbes, providing defence against viral infection. Small CRISPR RNAs encoded by the host genome are loaded into Effector complexes and used to detect and destroy invading viruses with similar sequences. This programmable "seek and destroy" system has recently been harnessed to direct the cleavage of specific gene targets in many organisms including human cells, and shows great promise in genome engineering and healthcare. The underlying basis for the CRISPR system is the capture of a library of small DNA fragments derived from invading viruses. The focus of this project is on the mechanism of "Adaptation", by which these DNA species are captured and integrated in the correct position in the host genome. Adaptation is very poorly understood with little mechanistic detail available. In this project the White (St Andrews) and Bolt (Nottingham) labs will combine their expertise in biochemistry and genetics to tackle this important question. The work will capitalise on some recent breakthroughs by both labs that highlight some key aspects of the Adaptation pathway. The work will lead to fundamental new insights into the Adaptation process and also pave the way towards biotechnological applications of the system.

CRISPR (clustered regularly interspaced palindromic repeats) is an adaptive, antiviral defence system found in prokaryotes. CRISPR loci in the genome store a record of past viral infection. Transcription of these loci yields CRISPR RNA (crRNA) that is loaded into large effector complexes and used to target and destroy invading mobile elements. One of these effector complexes, Cas9, has shown great utility in targeted genome engineering in many biological systems from bacteria and plants to human cells. The underlying basis for the whole CRISPR system is the ability to capture small pieces of invading viral DNA and incorporate them into the genome to provide a memory of past infections. This process, known as "Adaptation" or "Acquisition", requires the Cas1 and Cas2 proteins, but is not understood at a mechanistic level and is widely acknowledged as the most important aspect of the CRISPR system requiring further study. Adaptation can be broken down into two parts - the capture of foreign DNA and subsequent integration into the CRISPR locus. In this project the White (St Andrews) and Bolt (Nottingham) labs will combine their expertise in biochemistry and genetics to tackle the mechanistic basis for CRISPR Adaptation in two model systems: S. solfataricus and E. coli. Both labs have made key advances in this area, showing that Adaptation involves replication fork restart in vivo in E. coli (Nottingham) and that Cas1 is highly specific for the trans-esterification of stalled replication fork model substrates in vitro (St Andrews). The work proposed promises to unravel the mechanism of Adaptation using genetics, molecular biology and biochemical techniques. The mechanism of Adaptation is the last significant missing piece of the CRISPR puzzle.

Academic impact
Scientists working in academia and industry will benefit from the basic advances in our understanding of the mechanism of Cas1-mediated DNA integration. There is the potential for new tools allowing genetic manipulation in cells. The UK scientific community does not have a strong presence in the CRISPR field and the project brings two of the leading labs together.
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 PDRA employed on the grant will receive extensive training in a variety of disciplines spanning microbiology through biochemistry to biophysics and single molecule techniques. she will have access to the award winning "Gradskills" courses run by the University of St Andrews, which aim to provide a wide variety of transferrable skills.
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, but this is almost wholly focused on Cas9. The integration of new DNA sequences into a specific area in a genome is clearly of potential utility in a wide variety of organisms if the technology can be understood and harnessed. Once the mechanism of Cas1-mediated DNA integration is understood, the potential for important applications in molecular biology and biotechnology will become apparent.