Mechanism of Co-Transcriptional DNA and RNA Targeting by Type III CRISPR-Cas Systems

项目总结 原核生物使用一种名为CRISPR-CA的适应性免疫系统来防御噬菌体和其他移动的 遗传因素。这些系统从它们的入侵者那里获取短的DNA片段，作为之前 感染。他们用它们来产生CRISPR RNA，这些RNA与一个或多个CAS蛋白组装成 RNA引导的干扰复合体，在外源DNA或RNA中发现并切割互补靶标。 虽然大多数类型的CRISPR-CAS系统只针对一种类型的核酸，但III型CRISPR系统具有 据报道，它同时针对DNA和RNA。因此，III型系统的真靶核酸的同一性 仍然不为人知。据报道，一种针对体内转录活性位点的细菌III-A型系统， 这导致了一种假设，即DNA和RNA都存在的转录泡泡是 生理靶点。因此，由干扰复合体进行的目标识别可以涉及同时识别 无论是DNA还是RNA。为了检验这个模型，我们建议1)捕获一个与A-A类(CSM)结合的复合体 转录活性靶标，并使用冷冻电子显微镜来确定络合物的结构和 2)使用蛋白质-核酸结合和核酸裂解试验来确定RNA结合是否招募到 III型-A型(CSM)复合体与转录泡结合。这将为重新利用这些资源奠定基础。 检测转录活性基因座，并调节异源生物中基因表达的系统。

PROJECT SUMMARY Prokaryotes use an adaptive immune system called CRISPR-Cas to defend against phages and other mobile genetic elements. These systems acquire short pieces of DNA from their invaders as a genetic record of prior infections. They use these to produce CRISPR RNAs that are assembled with one or more Cas proteins into RNA-guided interference complexes that find and cleave a complementary target in foreign DNA or RNA. While most types of CRISPR-Cas systems target only one type of nucleic acid, Type III CRISPR systems have been reported to target both DNA and RNA. The identity of the true target nucleic acid of Type III systems thus remains unknown. A bacterial Type III-A system was reported to target transcriptionally active loci in vivo, leading to the hypothesis that the transcription bubble, at which DNA and RNA are both present, is the physiological target. Target recognition by the interference complex thus may involve simultaneous recognition of both DNA and RNA. In order to test this model, we propose to 1) trap a Type III-A (Csm) complex bound to a transcriptionally active target and use cryo-electron microscopy to determine the structure of the complex and 2) use protein-nucleic acid binding and nucleic acid cleavage assays to determine if RNA binding recruits the Type III-A (Csm) complex to the transcription bubble. This would lay the groundwork for repurposing these systems to detect transcriptionally active loci, and regulate gene expression in heterologous organisms.