Building CRISPR Immunity Systems - How is Invading DNA Captured?

CRISPR systems have evolved in microbes to give them immunity against death or unwanted genetic baggage from viruses and other mobile genetic elements (MGEs). The immunity system is built when fragments of MGE DNA are recognised, captured and stored in the microbe's CRISPR system - these processes are called "adaptation". Once stored, the MGE DNA fragments in CRISPR are converted into RNA by transcription, and the CRISPR RNA is used to seek and destroy returning MGE DNA, therefore protecting the microbial cell from re-infection and death.

Some parts of the processes that control CRISPR-based adaptation are known, however it is unknown how viral DNA/RNA is recognised as "non-self" and is therefore captured to establish immunity the first time it is encountered by the microbe. We know that Cas1-Cas2 enzyme complex is essential for CRISPR adaptation, but we do not know fully how adaptation is achieved either in natural cellular systems or in the molecular detail of individual genes and proteins.

We will investigate the cell and molecular biology of the Cas1-Cas2 enzyme complex to understand how it can capture fragments of virus DNA. This will be performed using E. coli as a model bacterium, examining the biochemistry of DNA capture, the genetic components that are vital parts of the process and using time lapse microscopic imaging of live cells to observe adaptation in real time and in unprecedented detail.

The new knowledge of how CRISPR immunity develops in bacteria is important for many different areas of biology, from microbiology and antibacterial resistance, DNA breaks and genome instability to the biotechnology applications of genetic engineering.

Understanding how immunity is generated in bacteria is important for microbiologists who are interested in antibiotic resistance as this is a challenge that urgently needs to be overcome. By knowing how CRISPR immunity functions in normal healthy bacteria will enable the development of natural strategies to overcome antibiotic resistance where the resistance genes are often carried on genetic elements that are destroyed by CRISPR.

Our new methods for imaging of Cas1 in cells will also benefit researchers interested in understanding genome dynamics in cells, specifically how and why DNA gets broken. This is directly relevant to biologists who wish to understand how genome instability arises and leads to the problems manifested in various human diseases such as cancer, and the ageing process.

CRISPR is widely used as biotechnology tool genetic engineering and editing in cells, but the Cas1-Cas2 complex is not as well developed as other CRISPR-based genetic editing methods e.g. Cas9. Understanding how Cas1-Cas2 can capture DNA molecules before storing them in a DNA fragment database e.g. CRISPR has potential to streamline its use as an editing tool in many applications.

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.

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 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.