Determining the specificity and regulation of Type III CRISPR-Cas interference

项目摘要 CRISPR（规律成簇间隔短回文重复序列）和CRISPR相关（Cas）蛋白 是一组不同的crRNA引导的核酸酶，为原核生物提供针对外源基因的适应性免疫， 元素Cas10-Csm是一种约300 kDa的多蛋白复合物， 转录物启动DNA和RNA降解反应。不同的CRISPR-Cas类型使用 crRNA检测双链DNA或单链RNA。虽然很多人都知道， 通过I型（Cascade）和II型（Cas9）系统的dsDNA检测的特异性机制，知之甚少 关于III型（Cas10）系统检测ssRNA的特异性机制。这是重要 因为有证据表明，在dsDNA检测和ssRNA检测过程中， 检测是根本不同的：非碱基配对的DNA链的再杂交之间的竞争 重新形成dsDNA或与Cas蛋白结合对于dsDNA检测特异性机制是必需的。这 在ssRNA的特异性检测过程中不可能存在这种机制。假设将被测试，在Cas10- crRNA-靶RNA双链体内特定敏感位置的CSM系统错配破坏 干扰Cas10-Csm结构模型表明Cas10-Csm的Csm 2组分直接接触 结合靶RNA。我们将检验Csm2在检测同源RNA中起着不可或缺的作用的假设 结合到Cas10-Csm并通过结构变化将该信号传递给Cas10。我们会用冷冻电镜 确定Cas10激活的“结构机制”。这项研究将影响几个 新兴的生物技术，如部署Cas10作为护理点RNA病毒诊断。

Project Summary CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins are a group of diverse crRNA guided nucleases providing prokaryotes adaptive immunity to foreign genetic elements. Cas10-Csm is an approximately 300 kDa multiprotein complex that upon detecting foreign RNA transcripts initiates a DNA and RNA degradation response. Distinct CRISPR-Cas types use the information in crRNA to detect either double-stranded DNA or single-stranded RNA. While much is known concerning the specificity mechanisms of dsDNA detection by Type I (Cascade) and Type II (Cas9) systems, little is known concerning the specificity mechanisms of ssRNA detection by Type III (Cas10) systems. This is significant because evidence exists that the mechanisms for enforcing specificity during dsDNA detection and ssRNA detection are fundamentally different: competition between rehybridization of the non-base paired DNA strand to re-form dsDNA or binding to a Cas protein is essential to the dsDNA detecting specificity mechanism. This mechanism is not possible during specific detection of ssRNA. The hypothesis will be tested that in the Cas10- Csm system mismatches in specific, sensitive locations within the crRNA-target RNA duplex disrupt interference. Cas10-Csm structural models suggest the Csm2 component of Cas10-Csm directly contacts bound target RNA. We will test the hypothesis that Csm2 plays an integral role in detecting cognate RNA binding to Cas10-Csm and relays this signal to Cas10 via structural changes. We will use cryo-EM to determine the `structural mechanism' for Cas10 activation. The research proposed will impact several emerging biotechnologies such as the deployment of Cas10 as a point-of-care RNA virus diagnostic.