Building CRISPR Immunity Systems - How is Invading DNA Captured?

Lead Research Organisation: Brunel University London
Department Name: Life Sciences


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Technical Summary

Genetic diversity in prokaryotes is stoked by gene flow between cells, enabled by mobile genetic elements (MGEs); viruses, transposons and plasmids. However, cell death and metabolic burden associated with MGEs has led to evolution of CRISPR immunity systems against them. We are investigating how CRISPR systems establish immunity against MGEs that have not been previously encountered - processes called "naïve adaptation".

The Cas1-Cas2 complex is essential for naïve adaptation, by capture of MGE DNA for integration into specific chromosomal sites called CRISPRs, which are the immune memory. Based on our new insights in published work and preliminary data, we hypothesise that DNA capture is achieved by Cas1-Cas2 nuclease activity at MGE DNA ends. Crucial to this is how Cas1-Cas2 achieves DNA target selection from a "non-self" MGE rather than "self" chromosomal DNA - we propose that this requires interaction of Cas1-Cas2 with the molecular chaperone DnaK, helped by the helicase activity of the RecBCD DNA repair protein.

We aim to deliver a mechanistic model for naïve adaptation that will greatly enable understanding of CRISPR systems. The project will utilise biochemistry, microscopy and genetics to delineate the mechanisms for DNA capture by Cas1-Cas2 targeted to MGE DNA. Understanding how CRISPR systems function in this way, and therefore how they become a barrier to gene flow in prokaryotic populations, is significant for understanding the spread of antibiotic resistance genes, and for the potential to exploit CRISPR systems to combat it.

A significant step forward in studying Cas1-Cas2 has arisen from our ability to visualise fluorescently tagged Cas1 in living cells that are responsive to the occurrence of dsDNA ends. This will provide new insights into CRIPSR adaptation but also into DNA break formation more generally. We also aim to develop this as a novel tool for studying dsDNA breaks, DNA repair and genome dynamics in living cells.

Planned Impact

By delineating mechanisms that establish CRISPR immunity by DNA capture we will generate new knowledge that immediately enables academic, medical and biotechnology researchers to interpret their data in new ways, and to develop new ideas. In the longer term, our research findings and development of our new methods from this project have potential to impact widely across cell biology and genome dynamics. In all cases we will ensure that our research findings, and ideas for their development, are disseminated widely through academic routes, opportunities to engage in technology transfer events, and by frequent outreach activities.

1. Short term direct impact:
1.1. The research groups of both co-applicants, and our collaborators at Zagreb University and Granada University, will benefit from knowledge exchange throughout the project that will provide them with state-of-the-art methods in CRISPR research and protein biochemistry that are relevant to their research in genome instability and genetic analysis of CRISPR systems.

1.2. Microbiologists, cell biologists and biotechnologists. Others can use our mechanistic model for DNA capture by Cas1-Cas2 for related research in understanding barriers to spread of antimicrobial resistance. The new knowledge will inform Cas1-based methods of genome editing, and help with interpretation of events in cells that trigger genome instability by DNA breakage.

1.3. Both early-career researchers (the PDRAs) will obtain cross-disciplinary training that will benefit their careers as scientists by enabling them to develop new experimental methods that can be deployed in their future careers. The project will encourage them to develop and re-enforce skills at outreach, research communication, project management and collaboration that will be valuable as they develop their careers. Both PIs take cohorts of PhD and MRes students, and undergraduate summer vacation students, who will also benefit from laboratory training, and will gain understanding of CRISPR systems, one of the hottest topics in molecular biology and biotechnology. This contributes to the UK research capacity and economy that is beneficial as a legacy for the future.

2. Longer term indirect impact:
2.1. a) Biotechnology and gene editing companies - these will benefit from the underpinning knowledge of how the Cas1-Cas2 complex functions to provide more streamlined tools for genetic manipulation.
b) the healthcare sector may benefit from understanding how bacteria protect themselves from MGEs that carry antimicrobial resistance genes and develop new strategies and therapeutic molecules to overcome antimicrobial resistance.

2.2. Ensuring UK leadership in CRISPR biotechnology, microbiology and cell biology - the training of early-career researchers, planned dissemination of the research and outreach activities will ensure that awareness of the project is spread to the widest possible audience, both nationally and internationally; building capacity and creating a recognisable hub of knowledge in the UK about CRISPR systems and how they can be applied in new technologies.

2.3 Benefits for quality of life and public health - society in general will benefit in the long term through improved knowledge of how DNA breaks occur in cells; this is important because DNA breaks cause genome instability that is an underlying mechanism for various diseases and health problems associated with ageing, including cancers. Therefore knowledge from the research project can enable healthcare researchers and providers to combat these kinds of diseases and health problems. Having new understanding for strategies and therapies that treat bacterial diseases currently caused by antibiotic resistant strains will also improve quality of life.


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Description The experimental work for this award was unfortunately massively impacted by the SARS-CoV-2 pandemic. Work started in February 2020 with preparatory experiments, which had to be halted early in March, first because of self-isolation and then because of the first National lockdown. Brunel University re-opened the laboratories in mid-September, and we had a fruitful 3 months of experimental work. Unfortunately work again needed to be halted because of the third national lockdown and the grant was suspended until April 2021. In addition, work had to be interrupted at various stages because of lab members or family members of lab workers catching COVID and needing to isolate. This means that since the beginning of the grant effectively 9 months of work by PI and postdoc were lost because of the pandemic.

Despite these difficulties we still have made significant progress. If over-expressed, fluorescently-labelled Cas1 protein (FP-Cas1) can be easily visualised in living cells and can bei tracked in time-lapse fluorescence microscopy experiments. Foci numbers are greatly increased if the chaperone DnaK is missing. This was a crucial finding, as it suggested that the chaperone DnaK might have a regulatory function for the CRISPR-Cas system. Especially the last 12 months were used for an intensely collaborative approach. The teams from Nottingham and Brunel met at least weekly for lab meetings and exchanged information. This has allowed us to demonstrate experimentally that DnaK and Cas1 interact both physically and functionally. In the presence of DnaK the acquisition of sequences into the CRISPR array is activly inhibited, while incorporation takes place faster if DnaK is missing, in line with our experimental result showing more foci throughout cells. Thus, our work has resulted in a model which predicts how cells can distinguish between foreign genetic material and its own chromosome. DnaK is localised effectively in spaces where the chromosome is located, preventing Cas1-Cas2 from acquiring substrate. However, if a bacteriophage such as phage lambda attacks, the chaperone DnaK is actively recruited to facilitate the inititation of phage DNA synthesis. This triggers a release of Cas1 and consequently the uptake of foreign sequences into the CRISPR array.

We have reported these intriguing findings in a manuscript, which is currently under evaluation with Nature Communications.
Exploitation Route We were able to demonstrate in E. coli that fluorescently-labelled Cas1-Cas2 complexes are a) binding a specific DNA substrate and b) only can be observed to do so if the cells are actively replicating. Fluorescent foci are observed in nearly all cells, suggesting that the intermediate bound is common in replicating cells. This opens the possibility that expression of fluorescently-labelled Cas1, together with Cas2, might be a marker for active DNA replication in cells other than E. coli, including other bacteria but also human cells. We are currently doing proof-of-principle experiments with our constructs, and we are in contact with the IP teams of both Universities to discuss a patent application.

Staff training: the award has already had significant impact. The postdoc for this study, Dr Dimude, has done a significant number of fluorescent microscopy experiments and has become much more familiar with the entire system. She is now performing sets of experiments completely independently and analyses the data, which was one of the outcomes we aimed for. We can now proceed to her to get familiar with more advanced experimental fluorescent analysis techniques required for this study.

By now (March 2023) several undergraduate work placement student have been trained in the lab, many of which have contributed to the project by doing molecular work. Thus, we have provided, and are still providing, significant training at undergraduate student level, providing students with experiences that include all aspects of lab work, from planning experiments, performing them and interpreting the associated data.

In addition, I had a PhD student who is funded by the College and who has started in the lab. We have started to do bioinformatics analysis both of E. coli genomes but also automated analysis of fluorescence microscopy images. This is an incredibly sought-after skill set. In fact, the Governmental Joint Security Centre was looking for PhD students with these skills and my PhD applied and got offered a part-time position to analyse SARS-CoV-2 variant genomes for a defined period of time, highlighting the impact the acquired skills are having, both for the researchers involved, but also (in this case) on a national level.
Sectors Education,Healthcare,Government, Democracy and Justice,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description We have provided experimental data that expressed CRISPR-Cas proteins will bind to a certain class of DNA intermediate that arises as DNA is replicated and repaired. While we are still working on establishing precisely what this DNA substrate is, we were able to show that in repair-deficient cells more binding is observed. For this we are using a fluorescent fusion protein that can be directly visualised. It is very likely that this will provide a tool for the direct visualisation of DNA repair/replication intermediates in other cells, and we are currently working on proof-of-principle experiments befpre an IP application can be considered.
First Year Of Impact 2021
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other
Impact Types Economic

Description Harry Smith Vacation Studentship 2020
Amount £2,720 (GBP)
Funding ID GA002310 
Organisation Microbiology Society 
Sector Learned Society
Country United Kingdom
Start 06/2020 
End 08/2020
Title BioID2 protein interaction mapping in E. coli 
Description Methods that identify protein-protein interactions are essential for understanding molecular mechanisms controlling biological systems. Proximity-dependent labelling has proven to be a valuable method for revealing protein-protein interaction networks in living cells. A mutant form of the biotin protein ligase enzyme from Aquifex aeolicus (BioID2) underpins this methodology by producing biotin that is attached to proteins that enter proximity to it. This labels proteins for capture, extraction, and identification. In this chapter we present a toolkit for BioID2 specifically adapted for use in E. coli, exemplified by the chemotaxis protein CheA. We have created plasmids containing BioID2 as expression cassettes for proteins (e.g., CheA) fused to BioID2 at either the N or C terminus, optimised with an 8 × GGS linker. We provide a methodology for expression and verification of CheA-BioID2 fusion proteins in E. coli cells, the in vivo biotinylation of interactors by protein-BioID2 fusions, and extraction and analysis of interacting proteins that have been biotinylated. 
Type Of Material Technology assay or reagent 
Year Produced 2023 
Provided To Others? Yes  
Impact No impacts yet. 
Description Ed Bolt 
Organisation University of Nottingham
Department School of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution My lab is actively working on generating strains in which proteins of the Cas-CRISPR system can be localised in vivo. We are investigating the role of the CRISPR-Cas system in bacteria in general and in E. coli in particular.
Collaborator Contribution Ed Bolt's lab is working on the in vitro reconstitution of the Cas-CRISPR system in Escherichia coli. The collaboration will allow us to combine both in vivo and in vitro approaches to characterise the system in E. coli and other bacterial organisms.
Impact No outputs yet
Start Year 2016
Description Ronan McCarthy 
Organisation Brunel University London
Department Division of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution My lab is providing a molecular biology of analysis of the effect of artificial sweeteners on cell growth and morphology, both E. coli and Acinetobacter baumannii
Collaborator Contribution Dr McCarthy is the PI of this particular project.
Impact Collaboration has led to a first high-profile publication in EMBO Molecular Medicine (
Start Year 2022
Description Thorsten Allers 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Exchange of research ideas and results.
Collaborator Contribution Prof. Allers is an expert in archaeal genomics and his input will allow us to work on comparative genomics of bacterial and archaeal chromosomes.
Impact No outcomes yet
Start Year 2021
Description CoolLED interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Based on an entry in blog of my internet page ( the company CoolLED reached out to me to conduct an interview about the research in my lab and how the recent accquisition of a pE-4000 LED illumination unit helps the work in my lab. This was published online and in their monthly newsletter, and links have been posted on Social Media such as Twitter.
Year(s) Of Engagement Activity 2021
Description Seminar Okinawa Institute of Science and Technology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I was invited to present the latest research of my group as a Research Seminar at the Okinawa Institute of Science and Technology, Graduate University, Onna, Okinawa, Japan. I was personally invited by Prof. Simone Pigolotti to give this research seminar. This meeting was attended both in person in via online connections by a large number of researchers from all over Japan and other locations. My talk sparked questions and discussions afterwards.
Year(s) Of Engagement Activity 2022
Description Seminar Tokyo Metropolitan 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I was invited to present the latest research of my group as a Research Seminar at the Institute of Medical Science, Tokyo Metropolitan University. I was personally invited by the Director of the Institute of Medical Science, Prof. Hisao Masai, to give this research seminar. This meeting was attended both in person in via online connections by a large number of researchers from all over Japan and other locations. My talk sparked questions and discussions afterwards.
Year(s) Of Engagement Activity 2022