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

项目概要/摘要： CRISPR和CRIPSR相关蛋白（Cas） 是具有记忆的细菌免疫系统的组成部分。Cas蛋白获得基因组的片段 并将它们放入CRISPR阵列中。在再感染时，Cas9或Cas 12 a被动员， 并装载有从CRISPR阵列转录的向导RNA。然后它们切割入侵的DNA链， 在产生一个20个碱基对的RNA-DNA异源双链体后， 一个R环R环的形成引发了两种酶中一系列复杂的构象变化， 通过途径上不同的检查点进行切割能力。重要的是这些 两种酶之间的构象变化不同，表明机理不同。可编程性 和由R环诱导的切割赋予的特异性使得Cas9和Cas 12 a成为优异的生物物理工具。 然而，这两种酶都可以结合并切割R环中具有错配的位点，导致 潜在危险的脱靶活动充分理解错配和靶DNA的影响 需要对R环形成和切割的拓扑结构进行优化以优化Cas9和Cas 12 a的使用和工程化。 细胞中的DNA在整体上是欠缠绕的，局部上是恒定的流动，以机械变形的过程 DNA.在这项提案中，布莱恩特实验室开发的高分辨率单分子方法将用于 观察Cas9/Cas 12 a R环形成和超螺旋DNA上的构象变化。 最近，这些方法被用于开发Cas9 R环形成的模型，其中R环错配 和DNA超螺旋改变了Cas9 R环形成能量景观的形状。的分辨率 方法允许鉴定离散的R环中间体。目前，类似的机型正在生产中 对于Cas 12 a，其也具有离散的R环中间体。这一提议的核心假设是， R环错配和DNA超螺旋调节Cas9/Cas 12 a 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。初步实验表明这些测量的可行性， 一致的R环和FRET信号。在目标2中，将使用Cas 12 a进行类似的测量。 目前的数据显示，Cas 12 a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将建立一个完整的图片，描述R环错配和DNA的影响， 超螺旋对Cas9和Cas 12 a活性和特异性的影响。从这些测量中发展出来的模型 将揭示Cas9/Cas 12 a机制和特异性差异之间的联系。这些信息将有助于 设计突变和扰动以最小化实验和临床环境中的脱靶活性。

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.