Mechanistic Studies of the Type I CRISPR-Cas system

项目摘要 原核生物可通过整合短片段获得对病毒和质粒的抵抗力 外来DNA，称为前起搏器，形成规则间隔的短回文重复序列 (CRISPR的)。然后这些重复序列被转录并加工成小的引导RNA 用来指挥对外来核酸的破坏。这种机制有许多相似之处 真核RNA干扰，但与CRISPR反应相关的蛋白质是 在进化上与它们的真核同行没有亲缘关系。我们的长期目标是了解 原核生物CRISPR介导抗性的生化和结构基础。目标 下面是为了了解靶序列的变化如何调节免疫反应 并提供有关如何将预定步器整合到CRISPR阵列中的详细信息。我们的 目标将结合结构、生化和基于细胞的实验。成功 拟议研究的完成具有重要意义，因为它将增加我们对 细菌对病毒和质粒的抵抗力，这两者在遗传学上都发挥着重要作用 致病细菌。这也是有意义的，因为这些研究将有助于进一步 开发基于CRISPR的工具。

Project Summary Prokaryotes can acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA, called prespacers, into clusters of regularly interspaced short palindromic repeats (CRISPR's). These repeats are then transcribed and processed into small guide RNA's that are used to direct the destruction of foreign nucleic acid. This mechanism has many parallels with eukaryotic RNA interference but the proteins that are associated with the CRISPR response are evolutionarily unrelated to their eukaryotic counterparts. Our long-term goal is to understand the biochemical and structural basis of CRISPR-mediated resistance in prokaryotes. The objectives here are to understand how changes in the target sequence modulate the immune response and provide insight into how the prespacers are incorporated into CRISPR arrays. Our objectives will combine structural, biochemical and cell based experiments. Successful completion of the proposed studies is significant because it will increase our understanding of bacterial resistance to viruses and plasmids, both of which play important roles in the genetics of pathogenic bacteria. It is also significant because these studies will help in the further development of CRISPR-based tools.