Microbial Warfare: Illuminating CRISPR adaptive immunity using single-molecule fluorescence

Bacteria and archaea are constantly threatened by a large array of viruses and other genetic elements. Driven by evolution, these organisms have acquired a wide arsenal of defense mechanisms that allow the host organism to fight off the invaders. Among these defense mechanisms is an adaptive and inheritable that is conveyed through Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR associated proteins (Cas). Immunity relies on the integration of short stretches of invasive DNA (spacers) into the genome of the host. Subsequent, transcription and processing of these spacers result in small crRNA molecules that guide Cas proteins for sequence specific DNA target degradation.
Most of our knowledge on CRISPR immunity has come from conventional biochemical techniques, that average the population dynamics and thereby mask the underlying molecular dynamics. In this thesis, we adopt single-molecule fluorescence techniques that allow for real-time visualization of the molecular dynamics that underlie CRISPR immunity. Our single-molecule approach reveals that the various stages of CRISPR immunity in E. coli are highly dynamic and tightly coupled, leading to a robust immune response. The results presented in this thesis contribute to a new level of understanding on the molecular mechanisms behind CRISPR immunity and may aid in the development of CRISPR-based tools for engineering biology.