Award details

CRISPR Adaptation - the basis for prokaryotic adaptive immunity

Reference	BB/M020541/1
Principal Investigator / Supervisor	Dr Edward Bolt
Co-Investigators / Co-Supervisors
Institution	University of Nottingham
Department	School of Life Sciences
Funding type	Research
Value (£)	328,715
Status	Completed
Type	Research Grant
Start date	31/08/2015
End date	19/10/2018
Duration	38 months

Abstract

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.

Summary

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.

Impact Summary

Pathways to Impact A. Academic beneficiaries We aim to elucidate mechanisms of CRISPR adaptation by Cas1-Cas2 proteins. The following academic and research groupings worldwide are likely to be beneficiaries: 1. Researchers in CRISPR immunity: The UK is under-represented in this. By combining our expertise, the Bolt and White labs aim to make advances with international impact. 2. Groups interested in protein machines that process nucleic acids (e.g. integrases). 3. Researchers in genome instability: We are investigating CRISPR adaptation linked to mechanisms of replication collapse and re-start. 4. Researchers of single molecule techniques with DNA/RNA processing enzymes: we have robust in vitro assays for spacer acquisition with potential for single molecule studies. Data arising from the project will be disseminated in international peer reviewed journals of high quality. We commit to presenting data at international conferences, including various CRISPR meetings and meetings on nucleic acid processing (e.g. Gordon Research meetings/Keystone/FASEB). The project includes collaboration with a non-UK research group (Zagreb, Croatia) that will promote UK-EU scientific partnership. B. Beneficiaries in the wider community The project can impact in the wider community by: (A) engagement of schools and general public, (B) commercialisation, and (C) collaborative training. (A). Schools, Communication and Public Engagement Research on CRISPR immunity is well suited for engagement of non-specialists, because it investigates viral attack, genome engineering and evolution, topics that can be presented imaginatively. I have been committed to public-engagement in science since 2002, and this will continue. (i) Since 2008 I have taught microbiology each year to children (7-11 years) at Nettleham Primary School, Lincolnshire. We do microbiology experiments related to DNA repair or food spoilage, with question and answers sessions. This is followed by a web-based quiz.The links I have made with the school are strong and will continue. I also give talks at schools (e.g. Durham School Scholar's dinner, March 2014 and Loughborough Grammar School, March 2013) and to other local organisations (e.g. Newark W.I.). These activities explain tax-payer funded research to the community, and enrich university teaching, by having to explain links between fundamental research, how it is reported in media, and where it can lead to in society. (ii). Until recently I was a volunteer working with visual artists within Ignite! and Creative Partnerships, organisations that promote science activities in schools: I worked in Garibaldi School, Mansfield. I will use the project described here to re-engage with my previous contacts for a new collaborative venture with visual artists. (B). Commercialisation Understanding Cas1-Cas2 mechanism during the project will be of interest to biotechnologists who are seeking ways to manipulate DNA using novel enzymes. A recent consultancy I undertook with Oxford Nanopore Technology opened possibilities to develop DNA processing activities for defined commercial purposes, and similar links will be sought for utilising especially Cas1, and possibly Cas2. Recently, The University of Nottingham has re-launched commercialisation operations, which include a consultancy unit within technology transfer and commercialisation. Through meetings with their representative I will develop this as the project develops. (C). Collaborative training and networking The project contains two collaborations, both already in place and both well primed for frequent movement of ideas between labs. I am especially alert to the fact that the project uses biochemistry, biophysics and genetics methods in focusing on one problem: this has potential to be a valuable training regime, or at least garnering new experience of research methods, for PDRAs and PhD students in all of the labs involved.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/M021017/1 CRISPR Adaptation - the basis for prokaryotic adaptive immunity

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