Prokaryotic Adaptive Immunity; Mechanisms and Regulation

值得注意的是，直到2008年，细菌和古细菌（原核生物）才被证明具有适应性免疫系统，可以保护它们免受原核病毒的侵害。这个系统被称为CRISPR/Cas，它使用基于蛋白质和RNA的识别来识别和摧毁入侵的病毒。然而，我们仍然缺乏关于如何实现这一目标的重要细节。该项目将利用生化方法进一步了解CRISPR/Cas系统，以及识别和破坏入侵病毒DNA所需的详细生化步骤。它还将研究控制这种免疫系统活动的方式。虽然目标是对CRISPR/Cas的生物学和化学有基本的生物学理解，但该系统识别、修复、改变或破坏活细胞中特定DNA序列的能力在生物技术中具有许多潜在的强大应用。最后，该项目与蒙大拿州立大学和国家科学基金会的教育目标紧密结合。高中、本科和博士阶段的学生（和教师）将接受生物化学、结构生物学和热生物学方面的重要培训。研究人员将利用多种生物物理和生化方法，包括x射线晶体学、单粒子低温电子显微镜、质谱和表面等离子体共振来完成该项目的具体目标。这些方法将首先用于了解CRISPR/Cas如何识别和模板CRISPR RNA以识别和破坏入侵的病毒DNA。这些相同的方法将用于研究Cascade的活性是如何被调节的。总之，这些研究将在原子水平上阐明400 kDa级联复合物的结构，确定控制其活性的翻译后修饰，并确定被认为调节CRISPR/Cas系统表达的分子。

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.