Building CRISPR Immunity Systems - How is Invading DNA Captured?

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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.