Remarkably, it was not until 2008 that Bacteria and Archaea (prokaryotes) were shown to possess adaptive immune systems that protect them against prokaryotic viruses. This system, called CRISPR/Cas, uses protein and RNA based recognition to identify and destroy an invading virus. However, we still lack important details on how this is accomplished. The project will utilize biochemical methods to further our understanding of the CRISPR/Cas system and the detailed biochemical steps it takes to recognize and destroy the invading viral DNA. It will also investigate the ways in which the activity of this immune system is controlled. Although the goal is a basic biological understanding of the biology and chemistry of CRISPR/Cas, the ability this system to recognize, repair, alter, or destroy a specific DNA sequence in a living cell has a host of potentially powerful applications in biotechnology. Finally, there is a strong integration of the project with the educational goals of Montana State University and the National Science Foundation. Students (and teachers) at the high school, undergraduate and doctoral levels will receive significant training in biochemistry, structural biology and thermal biology.
The investigators will utilize a number of biophysical and biochemical methods, including X-ray crystallography, single particle cryo-electron microscopy, mass spectrometry and surface plasmon resonance to accomplish the specific goals of this project. These methods will first be used to understand how the CRISPR/Cas recognizes and templates CRISPR RNA for the recognition and destruction of invading viral DNA. These same methods will be used to investigate how the activity of Cascade is regulated. Combined, these studies will elucidate the structure of the 400 kDa Cascade complex at the atomic level, identify post-translational modifications that control its activity and identify molecules thought to regulate the expression of the CRISPR/Cas system.
