Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
Department of Statistics, Iowa State University, Ames, IA 50011, USA.
Cell Rep. 2017 Dec 26;21(13):3717-3727. doi: 10.1016/j.celrep.2017.11.110.
CRISPR-Cas systems defend bacteria and archaea against infection by bacteriophage and other threats. The central component of these systems are surveillance complexes that use guide RNAs to bind specific regions of foreign nucleic acids, marking them for destruction. Surveillance complexes must locate targets rapidly to ensure timely immune response, but the mechanism of this search process remains unclear. Here, we used single-molecule FRET to visualize how the type I-E surveillance complex Cascade searches DNA in real time. Cascade rapidly and randomly samples DNA through nonspecific electrostatic contacts, pausing at short PAM recognition sites that may be adjacent to the target. We identify Cascade motifs that are essential for either nonspecific sampling or positioning and readout of the PAM. Our findings provide a comprehensive structural and kinetic model for the Cascade target-search mechanism, revealing how CRISPR surveillance complexes can rapidly search large amounts of genetic material en route to target recognition.
CRISPR-Cas 系统可抵御噬菌体和其他威胁对细菌和古菌的感染。这些系统的核心组件是监视复合物，它使用指导 RNA 来结合外来核酸的特定区域，将其标记为待破坏的目标。监视复合物必须快速定位目标，以确保及时的免疫反应，但搜索过程的机制仍不清楚。在这里，我们使用单分子 FRET 实时可视化 I-E 型监视复合物 Cascade 如何搜索 DNA。Cascade 通过非特异性静电接触快速且随机地对 DNA 进行采样，在短的 PAM 识别位点处暂停，这些识别位点可能与目标相邻。我们确定了对于非特异性采样或定位以及 PAM 读取至关重要的 Cascade 基序。我们的研究结果为 Cascade 目标搜索机制提供了一个全面的结构和动力学模型，揭示了 CRISPR 监视复合物如何在识别目标的过程中快速搜索大量遗传物质。
