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

项目摘要 CRISPR(簇状规则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，为原核生物提供对外来基因的适应性免疫。 元素。Cas10-CSM是一种约300 kDa的多蛋白复合体，当检测到外源RNA时 转录本启动DNA和RNA降解反应。不同的CRISPR-CAS类型使用中的信息 CrRNA用于检测双链DNA或单链RNA。虽然关于这个问题我们知道的很多 I型(Cascade)和II型(Cas9)系统检测dsDNA的特异性机制，目前知之甚少 关于III型(Cas10)系统检测单链RNA的特异性机制。这一点意义重大 因为有证据表明，在dsDNA检测和单链RNA检测过程中实施特异性的机制 检测是根本不同的：非碱基配对DNA链的重新杂交之间的竞争 重组dsDNA或与Cas蛋白结合是dsDNA检测特异性机制的关键。这 在特异性检测单链RNA的过程中，机制是不可能的。这一假设将得到检验，即在Cas10- 在crRNA-靶RNA双链中断内特定敏感位置的CSM系统不匹配 干扰。Cas10-CSM结构模型表明Cas10-CSM的CSM2组件直接接触 结合的靶向RNA。我们将检验这一假设，即Csm2在检测同源RNA中起着不可或缺的作用 与Cas10-CSM结合，并通过结构变化将该信号传递给Cas10。我们将使用冷冻-EM来 确定Cas10激活的“结构机制”。这项拟议的研究将影响几个 新兴生物技术，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.