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

项目摘要/摘要： 簇状规则间隔短回文重复序列(CRISPR)和CRIPSR相关蛋白(Cas) 是具有记忆力的细菌免疫系统的组成部分。CAS蛋白获得基因组片段 并将它们放入CRISPR阵列中。在再次感染时，Cas9或Cas12a被动员 并装载有转录自CRISPR阵列的引导RNA。然后它们裂解入侵的DNA链， 含有与引导RNA匹配的序列后，创建了20个碱基对的RNA-DNA异源双链 一个R环。R-环的形成在两种酶中启动了一系列复杂的构象变化，每种酶 在切割能力的道路上通过不同的检查点。重要的是，这些 这两种酶的构象位移不同，表明其机理不同。可编程性 R环诱导的切割所赋予的特异性使Cas9和Cas12a成为优秀的生物物理工具。 然而，这两种酶都可以结合和切割在R环中具有错配的位置，从而导致 潜在危险的偏离目标的活动。充分了解错配和靶DNA的影响 为了优化Cas9和Cas12a的使用和工程，需要关于R-环形成和切割的拓扑。 细胞中的DNA是整体缠绕的，局部处于恒定的流量下，以机械变形的方式进行应有的过程 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被用于 在超螺旋DNA上同时观察Cas9/Cas12a R环的形成和构象变化。 最近，这些方法被用来开发一个R-环不匹配的Cas9 R-环形成模型 DNA超螺旋改变了Cas9 R环形成能量格局的形状。的解决方案 方法可以鉴定一个离散的R环中间体。目前，一种类似的模型正在生产中 对于Cas12a，它也有一个离散的R环中间体。这项提议的中心假设是 R-环失配和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-LOOP状态， 关联构象检查点和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，这些测量是可行的，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。 目前的数据显示，Cas12a具有不同的R环检查点，对超线圈高度敏感。数据 在这些目标中获得的将构建描述R环错配和DNA影响的完整图景 超螺旋对Cas9和Cas12a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/Cas12a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，以最大限度地减少实验和临床环境中的偏离目标的活动。

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.