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

项目摘要/摘要 许多人类疾病是由扰乱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用 基因组编辑，并在动物模型中研究结果。规则间隔成簇的回文重复 (CRISPR)相关蛋白，如Cas9，已经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，它使用起来更简单。Cas9是一个 源自原核生物的核酸内切酶，被引导到基因组中的20个核苷酸序列，称为 引导RNA的Protspacer。可以通过改变引导RNA的序列来对Cas9进行编程 一种可编程DNA切割蛋白。这项技术已经被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，低效率的活动在 哺乳动物细胞是阻碍广泛使用的瓶颈。Cas9特异性增强变异体和活性 为了提高活性，已经开发了优化和引导的RNA。我们和其他组织研究了如何 Cas9变异体和不同的引导RNA影响DNA的结合、解离和切割。尽管 正确的Cas9复合体组装、Cas9复合体结合和Cas9对DNA切割的重要性 染色质致密的哺乳动物细胞，我们对这些过程和分子细节的理解是 可怜。在两个具体目标中，我建议通过首先量化和真实观察来填补这些知识空白-- Cas9复合体如何组装的时间，以及它的核小体活性。第一个目标是适应以前的 建立了单分子实验，我可以模拟共转录的RNA折叠来研究分子 Cas9在折叠时与引导RNA组装的步骤。目标二将探讨Cas9如何结合和切割 使用高通量测序分析，DNA作为核小体周围DNA灵活性的函数。那我会的 量化结合动力学和平衡常数作为核小体周围DNA柔韧性的函数。 这些实验的结果将大大有助于我们对CRISPR CA的基本理解 并将帮助开发基于Cas9的癌症和神经退行性疾病疗法。 实现这些目标还将提供多色单分子FRET、生物化学、 分子生物学和下一代测序。此外，分析数据，撰写稿件 总结我的发现，并在会议上发表演讲将增强量化和软技能。《Ha》 实验室和约翰霍普金斯大学是进行这种研究培训的良好环境，主要是因为 获得广泛的专门知识和资源的机会。

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.