CRISPR Adaptation - the basis for prokaryotic adaptive immunity

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description The research funded was to understand how bacteria use specialised 'CRISPR systems' to identify and capture DNA molecules that are invasive (e.g. alien or 'non-self') - they do this using an enzyme complex called Cas1-Cas2. When Cas1-Cas2 captures non-self DNA it precisely inserts it into a specialised chromosomal site called a 'CRISPR' locus. This is the basis of an immunity system in bacteria that is effective against non-self DNA - CRISPR is a library of prior DNA invasions that can be activated into RNA for targeting to subsequent invading DNA, which destroys it.
Our research investigated this DNA capture step, because though essential for CRISPR immunity it is not understood. In particular there is no consensus about how DNA repair enzymes in cells help Cas1-Cas2 for capture. Our funded research was able to demonstrate two key findings; 1. That the paradigm for RecBCD nuclease targeting and generating invader DNA for capture is wrong. 2. That interactions of Cas1-Cas2 as a nuclease at DNA ends is crucial for DNA capture. We were therefore able to generate a new model for DNA capture in CRISPR systems and refurte previous high-profile data that is flawed.
Exploitation Route In two ways: First, we have over-hauled the prevailing model (published by others in Nature in 2014, Levy et al) for how CRISPR adaptation occurs in relation to RecBCD - work related to that is now funded as a new BBSRC grant (BB/T006625/1). Second, we have generated a new genetic editing tool as a spin-off from this research - this will be appearing as a new research publication in 2020.
Sectors Agriculture, Food and Drink,Creative Economy,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description 1. Internationally recognised research in the area of genome editing, which we have explained to the public in events such as Nottingham's festival of Science, and at schools. 2. Generation of a new biotechnology tool (protection being sought currently - publication expected in 2020). This has potential for use by others and will be deposited in AddGene. 3. I was consulted, for expertise on CRISPR systems and their uses, by The Biochemical Society for their submission on genetic editing to a House of Parliament committee, and by The BioIndustry Association for comments on a draft questionnaire to be circulated to their members to compile evidence for consideration by the European Commission in its CRISPR legislation.
First Year Of Impact 2018
Sector Communities and Social Services/Policy,Creative Economy,Culture, Heritage, Museums and Collections,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural,Societal,Economic,Policy & public services

 
Description DTP BBSRC
Amount £20,000 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 05/2017 
End 09/2020
 
Description CRISPR-Archaea 
Organisation Tel Aviv University
Country Israel 
Sector Academic/University 
PI Contribution Expertise in CRISPR-Cas
Collaborator Contribution genetic analysis and provision of starnds and DNA
Impact In preparation:Grant application to Human Frontier Science Program involving 4 teams of scientists, inlcuding myself.
Start Year 2014
 
Description CRISPR-Archaea 
Organisation University of Regensburg
Country Germany 
Sector Academic/University 
PI Contribution Expertise in CRISPR-Cas
Collaborator Contribution genetic analysis and provision of starnds and DNA
Impact In preparation:Grant application to Human Frontier Science Program involving 4 teams of scientists, inlcuding myself.
Start Year 2014
 
Description CRISPR-Archaea 
Organisation University of Ulm
Country Germany 
Sector Academic/University 
PI Contribution Expertise in CRISPR-Cas
Collaborator Contribution genetic analysis and provision of starnds and DNA
Impact In preparation:Grant application to Human Frontier Science Program involving 4 teams of scientists, inlcuding myself.
Start Year 2014
 
Description Discuva 
Organisation Discuva Ltd
Country United Kingdom 
Sector Private 
PI Contribution Research Processing and Analysis
Collaborator Contribution Provision of a high throughput library for analysis
Impact Ongoing
Start Year 2015
 
Description Live Cell Microscopy of Cas enzymes and replication forks in bacteria 
Organisation Brunel University London
Department Department of Psychology
Country United Kingdom 
Sector Academic/University 
PI Contribution The molecular model. Clones. Technical development
Collaborator Contribution Microscopy
Impact Preliminary data for a follow on grant
Start Year 2016
 
Description Reconstitution in vitro of CRISPR immunity and DNA replication 
Organisation University of York
Department Department of Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution The experiments are being developed to test biochemically the dynamics of interaction between CRISPR interference, adaptation and DNA replication. This requires us to do reconstitution of CRISPR interference and adaptation reactions, that will be used in DNA replication reactions.
Collaborator Contribution They provide a fully reconstituted DNA replication system for combining with CRISPR interference and adaptation assays.
Impact Further grant funding. Publications.
Start Year 2016
 
Description CRISPR "the magic scissors" at Nottingham Festival of Science and Curiosity, organised by Ignite! 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact An interactive display of CRISPR-Cas9 genome editing carried out at the Ignite! Festival of Science and Curiosity. The event was arranged in a section of the main Nottingham public library and leafleted in the weeks beforehand electronically and at various outlets in Nottingham city.
Year(s) Of Engagement Activity 2019
URL http://nottsfosac.co.uk/
 
Description Future Now Public Lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A talk and Q&A with the general public about CRISPR genome editing.
Year(s) Of Engagement Activity 2018
 
Description Invited seminar (University of Lancaster) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Invited seminar speaker
Year(s) Of Engagement Activity 2017
 
Description Research Talk, Berlin 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited Talk: Berlin
Year(s) Of Engagement Activity 2017
 
Description STEM lecture: School Visit (Denbigh, Milton Keynes) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Invited talk on CRISPR engineering for the after school public lectures series
Year(s) Of Engagement Activity 2017
URL http://www.denbigh.net/2014%20Pages/StemLectures/StemLectures.html
 
Description School Visit Nettleham, Lincs 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Microbiology talk and workshop to classes of children at a school for key stage 3 of the National Curriculum Science
Year(s) Of Engagement Activity 2015
 
Description University of Nottingham Suppliers Day talks x 2 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Talk on CRISPR to suppliers, companies and technical staff that supply The Univerisyt of Nottingham. OIne talk at The Sutton Bonnington Campus, the other at The University Park Campus
Year(s) Of Engagement Activity 2016