Periodic Reporting for period 4 - REMEMBER (Adaptive immunity in prokaryotes: how Bacteria do not forgive and do not forget their enemies) Reporting period: 2019-09-01 to 2020-05-31 Summary of the context and overall objectives of the project Microbes in natural ecosystems are under constant evolutionary pressure from viruses. To survive in this hostile environment microbes have evolved an adaptive immune system called CRISPR-Cas. The REMEMBER project set out to determine the mechanism of the enigmatic process of memory formation in CRISPR against mutated viruses that infect bacteria. Using a combination of genetic, biochemical and structural approaches, we have investigated the hypothesis that perfectly matching and degenerate targets trigger simultaneous virus DNA degradation and memory updates. We have also tested how CRISPR systems prevent autoimmunity: how do bacteria prevent accidental targeting of themselves, with lethal consequences as a result. The knowledge acquired will help us develop better strategies to use bacteriophages as alternatives for antibiotics. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Project REMEMBER has focused on how CRISPR systems keep their memory up to date. Nowadays CRISPR is synonymous with genome editing with Cas9, but CRISPR is much more complex and sophisticated than that. Bacteria immunize themselves against viruses using CRISPR and this step is not well understood. You may consider this step to be comparable to a vaccination in which our bodies are exposed to weakened viruses to trigger an immune response that protects us from infection by a real virus. The challenge for immune systems, however, is to deal with ever changing viruses. We found out that CRISPR can update its immunological memory by coupling inefficient immune reaction to new immunizations. This way the response will be amplified and the bacterium keeps track of the ever-mutating virus.Project REMEMBER has made big steps to achieve a solid understanding of the memory formation process in CRISPR-Cas systems. Compared to 5 years ago, the memory formation process is much better understoond. For example we now understand the role of the enigmatic protein Cas4 in memory formation, where it makes sure that a critical DNA sequence called the PAM is selected. This will allow a functional CRISPR response. Furthermore, project remember has uncovered the intimate link between target degradation and the formation of DNA substrates forming CRISPR memories. In addition, we have observed and measured how fast CRISPR complexes carry out their DNA surveillance in the cell in search for mutated and completely matching targets. This finding explains how CRISPR-Cas systems can be incredibly efficient.Lastly we have uncovered how the orientation of of integration of new CRISPR memories in the bacterial genome is achieved. All in all, project REMEMBER has been a great success. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The project has yielded many results reported in over 20 scientific publications and public lectures and public outreach. The results allow us to predict if bacteria will rapidly become resistant to bacteriophages when used in potential therapeutic applications. More clinical research is planned to further the project and allow the development of alternatives to antibiotics. phages-and-crispr-in-action-flyer.jpg
