Multimodal Single-molecule Analysis of DNA Interrogation by Cas9 and Cas12a: Examining the relationship between mismatches, DNA supercoiling, and conformational dynamics

Cas9 和 Cas12a 对 DNA 询问的多模式单分子分析：检查错配、DNA 超螺旋和构象动力学之间的关系

• 批准号: 10597025
• 负责人: Kevin Aris
• 金额: $ 4.02万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10597025
• 关键词: Base Pairing; Binding; Biophysics; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Competence; Complex; Coupled; Coupling; DNA; DNA analysis; Data; Disease; Due Process; Engineering; Enzymes; Event; Fluorescence Resonance Energy Transfer; Genetic Engineering; Genetic Transcription; Genomic Segment; Geometry; Guide RNA; Heteroduplex DNA; Immune system; Infection; Invaded; Kinetics; Label; Link; Magnetism; Measurement; Measures; Mechanics; Memory; Methods; Microscopy; Modeling; Molecular Conformation; Motion; Mutation; Pathway interactions; Predisposition; Process; Property; Protein Conformation; Proteins; RNA; Reporting; Research; Resolution; Risk; Role; SET Domain; Sampling; Shapes; Signal Transduction; Site; Specificity; Speed; Stress; Structure; Superhelical DNA; Technology; Testing; Therapeutic; Time; Torque; Update; Visualization; Work; biological research; biophysical tools; checkpoint modulation; clinical application; conformational conversion; design; endonuclease; experimental study; improved; insight; kinetic model; minimal risk; multimodality; nuclease; particle; pathogen; precise genome editing; single molecule; single-molecule FRET; spatiotemporal; tool

Project Summary/Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR) and CRIPSR associated (Cas) proteins are components of a bacterial immune system with memory. Cas proteins acquire segments of the genomes of invading pathogens and place them in the CRISPR array. Upon reinfection, Cas9 or Cas12a are mobilized and loaded with guide RNAs transcribed from the CRISPR array. They then cleave invading DNA strands that contain sequences matching the guide RNA after the creation of a 20-base pair RNA-DNA heteroduplex called an R-loop. R-loop formation initiates a complex set of conformational shifts in both enzymes, with each proceeding through distinct checkpoints on the pathway to cleavage competency. Importantly, these conformational shifts differ between the two enzymes, indicating mechanistic differences. The programmability and specificity imparted by R-loop induced cleavage make Cas9 and Cas12a excellent biophysical tools. However, both enzymes can bind to and cleave sites that possess mismatches in the R-loop, leading to potentially hazardous off-target activity. A full understanding of the effect of mismatches and target DNA topology on R-loop formation and cleavage is needed to optimize usage and engineering of Cas9 and Cas12a. DNA in cells is globally underwound and locally under constant flux to due processes that mechanically deform DNA. In this proposal, high-resolution single-molecule methods developed in the Bryant lab will be used to observe Cas9/Cas12a R-loop formation and conformational changes simultaneously on supercoiled DNA. Recently, these methods were used to develop a model for Cas9 R-loop formation in which R-loop mismatches and DNA supercoiling alter the shape of the Cas9 R-loop formation energy landscape. The resolution of the methods allowed identification of a discrete R-loop intermediate. Currently, a similar model is being produced for Cas12a, which also has a discrete R-loop intermediate. The central hypothesis of this proposal is that R-loop mismatches and DNA supercoiling modulate kinetic transitions between Cas9/Cas12a R-loop and conformational checkpoints. In aim 1, Cas9 FRET and R-loop states will be simultaneously observed, correlating conformational and R-loop checkpoints. This will require technical updates to microscopy methods to increase resolution. Preliminary experiments indicate the feasibility of these measurements, showing coincident R-loop and FRET signals. In aim 2, similar measurements will be performed using Cas12a. Current data show that Cas12a has different R-loop checkpoints and is highly sensitive to supercoiling. Data acquired in these aims will build a complete picture describing the effect of R-loop mismatches and DNA supercoiling on Cas9 and Cas12a activity and specificity. The models developed from these measurements will reveal links between Cas9/Cas12a mechanistic and specificity differences. This information will assist in designing mutations and perturbations to minimize off-target activity in experimental and clinical settings.

项目摘要/摘要： 成簇的规则间隔短回文重复序列 (CRISPR) 和 CRIPSR 相关 (Cas) 蛋白 是具有记忆的细菌免疫系统的组成部分。 Cas蛋白获取基因组片段 入侵病原体并将其放入 CRISPR 阵列中。再感染后，Cas9 或 Cas12a 被激活 并装载有从 CRISPR 阵列转录的引导 RNA。然后它们切割入侵的 DNA 链， 包含在创建 20 碱基对 RNA-DNA 异源双链体后与指导 RNA 匹配的序列，称为 R 环。 R 环的形成引发了两种酶中一系列复杂的构象转变，每种酶都发生了构象转变。 在通往裂解能力的道路上经历不同的检查点。重要的是，这些 两种酶之间的构象变化不同，表明存在机制差异。可编程性 R 环诱导切割带来的特异性使 Cas9 和 Cas12a 成为出色的生物物理工具。 然而，这两种酶都可以结合并切割 R 环中具有错配的位点，从而导致 潜在危险的脱靶活动。全面了解错配和目标 DNA 的影响 需要 R 环形成和裂解的拓扑结构来优化 Cas9 和 Cas12a 的使用和工程。 细胞中的 DNA 整体处于缠绕状态，并且局部处于恒定的流动状态，以适应机械变形的过程 脱氧核糖核酸。在该提案中，布莱恩特实验室开发的高分辨率单分子方法将用于 观察超螺旋 DNA 上 Cas9/Cas12a R 环的形成和构象变化。 最近，这些方法被用来开发 Cas9 R 环形成模型，其中 R 环不匹配 DNA 超螺旋改变了 Cas9 R 环形成能量景观的形状。该决议的 方法允许鉴定离散的 R 环中间体。目前，类似型号正在生产中 Cas12a 也有一个离散的 R 环中间体。该提案的中心假设是 R 环错配和 DNA 超螺旋调节 Cas9/Cas12a R 环之间的动力学转变 和构象检查点。在目标 1 中，将同时观察 Cas9 FRET 和 R 环状态， 关联构象和 R 环检查点。这将需要对显微镜方法进行技术更新 以提高分辨率。初步实验表明了这些测量的可行性，表明 重合的 R 环路和 FRET 信号。在目标 2 中，将使用 Cas12a 进行类似的测量。 目前的数据表明Cas12a具有不同的R环检查点并且对超螺旋高度敏感。数据 在这些目标中获得的知识将构建一幅完整的图景，描述 R 环错配和 DNA 的影响 超螺旋对 Cas9 和 Cas12a 活性和特异性的影响。根据这些测量结果开发的模型 将揭示 Cas9/Cas12a 机制和特异性差异之间的联系。此信息将有助于 设计突变和扰动以尽量减少实验和临床环境中的脱靶活动。