Mechanistic Studies of the Type I CRISPR-Cas system

项目摘要 原核生物可以通过整合短片段来获得对病毒和质粒的抗性。 外来DNA，称为prespacers，成簇的定期间隔短回文重复 （CRISPR）。然后这些重复序列被转录并加工成小的指导RNA， 用于直接破坏外源核酸。这一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 在进化上与真核生物无关。我们的长期目标是了解 CRISPR介导的原核生物抗性的生物化学和结构基础。目标 这里是为了了解靶序列的变化如何调节免疫反应， 并提供如何将预定距器并入CRISPR阵列的见解。我们 目标将结合联合收割机结构，生物化学和细胞为基础的实验。成功 完成拟议的研究是重要的，因为它将增加我们对 细菌对病毒和质粒的抗性，这两者在遗传学中起着重要作用 致病菌的数量这也很重要，因为这些研究将有助于进一步研究 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.