Direct observation and quantification of the assembly of Cas9 ribonucleoprotein complex and its activity on nucleosomes at single molecule resolution

项目总结/摘要 许多人类疾病都是由破坏细胞正常行为的突变引起的，例如癌症和 神经退行性疾病研究基因功能的一种方法是使用 基因组编辑并在动物模型中研究结果。规则间隔的重复序列 （CRISPR）相关蛋白，如Cas9，已成为两者的首选基因组编辑工具。 医疗保健和生命科学应用，因为与其他方法相比，它使用简单。Cas9是一个 来自原核生物的核酸内切酶，其被引导至基因组中的20个核苷酸序列，称为 原型间隔区通过引导RNA。Cas9可以通过改变引导RNA的序列来编程， 一种可编程的DNA切割蛋白这项技术已经广泛应用于生命科学中的基因研究 第一批基于Cas9的药物正在进入1期临床试验。然而，效率低下的活动， 哺乳动物细胞是阻碍广泛使用的瓶颈。Cas9特异性增强的变体和活性 已经开发了优化的和指导的RNA来提高活性。我们和其他组织研究了 Cas9变体和不同的指导RNA影响DNA结合、解旋和切割。尽管 适当的Cas9复合物组装、Cas9复合物结合和Cas9切割DNA的重要性 染色质致密的哺乳动物细胞，我们对这些过程的分子细节的理解， 扶贫在两个具体的目标中，我建议首先通过量化和观察真实的来填补这些知识空白- 时间Cas9复合物如何组装，以及其核小体的活性。目标之一是适应以前 我开发了单分子测定，在那里我可以模拟共转录RNA折叠来研究分子 Cas9与引导RNA组装的步骤，因为它折叠。目标二将探索Cas9如何结合和切割 使用高通量测序测定的DNA作为核小体周围DNA柔性的函数。然后我将 将结合动力学和平衡常数定量为核小体周围DNA柔性的函数。 这些实验的结果将大大有助于我们对CRISPR Cas的基本理解。 这将有助于开发用于癌症和神经退行性疾病的基于Cas9的疗法。 实现这些目标还将提供单分子FRET，生物化学， 分子生物学和下一代测序。此外，分析数据，撰写手稿 总结我的发现，并在会议上发表，将提高量化和软技能。医管局 实验室和约翰霍普金斯大学是这种研究培训的良好环境，主要是因为 获得广泛的专业知识和资源。

Project Summary/Abstract Many human diseases arise from mutations that disrupt the cell’s normal behavior, such as in cancer and neurodegenerative disorders. One method to study the function of genes is to ablate their function using genome editing and study the outcome in animal models. Clustered regularly interspaced palindromic repeats (CRISPR) associated proteins, such as Cas9, have emerged as the preferred genome editing tool for both healthcare and life science applications because it is simple to use compared to other methods. Cas9 is an endonuclease derived from prokaryotes that is guided to a 20-nucleotide sequence in the genome called the protospacer by a guide RNA. Cas9 can be programmed by changing the sequence of the guide RNA making it a programmable DNA cutting protein. This technology is already widely used in the life sciences to study gene function, and the first Cas9 based drugs are entering phase-1 clinical trials. However, inefficient activity in mammalian cells is a bottleneck preventing widespread usage. Cas9 specificity enhanced variants and activity optimized and guide RNA have been developed to improve activity. We and other groups have studied how Cas9 variants and different guide RNAs influence DNA binding, unwinding and cleavage. Despite the importance of proper Cas9 complex assembly, Cas9 complex binding and Cas9 cleavage of DNA in a chromatin compacted mammalian cell, our understanding of the molecular details of these processes and is poor. In two specific aims, I propose to fill in these knowledge gaps by first quantifying and observing in real- time how Cas9 complex assembles, and its activity of nucleosomes. Aim one is to adapt a previously developed single molecule assay where I can mimic co-transcriptional RNA folding to studying the molecular steps of Cas9 assembly with the guide RNA as it folds. Aim two will probe how Cas9 can binds and cleaves DNA as a function of DNA flexibility around nucleosomes using a high-throughput sequencing assay. I will then quantify the binding kinetics and equilibrium constants as a function of DNA flexibility around nucleosomes. The results of these experiments will significantly contribute to our fundamental understanding of CRISPR Cas enzymes and will aid efforts to develop Cas9 based therapeutics for cancers and neurodegenerative diseases. Accomplishing these aims will also provide technical training in multicolor single molecule FRET, biochemistry, molecular biology and next-generation sequencing. Furthermore, analyzing data, writing manuscripts summarizing my findings, and presenting at conferences will enhance quantification and soft skills. The Ha laboratory and Johns Hopkins University are excellent environments for this research training mainly because of the access to a broad range of expertise and to resources.