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

单分子分辨率下直接观察和定量 Cas9“核糖核蛋白复合物”的组装及其对核小体的活性

• 批准号: 10224792
• 负责人: Ikenna Okafor
• 金额: $ 4.6万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2022-06-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10224792
• 关键词: Adenine; Adopted; Affect; Affinity; Animal Model; Base Sequence; Basic Science; Behavior; Binding; Biochemistry; Biological Assay; Biological Sciences; Biotin; Blindness; CRISPR therapeutics; Cells; Chromatin; Cleaved cell; Color; Complex; Cytosine; DNA; DNA Binding; DNA Sequence; DNA-Protein Interaction; Data; Data Analyses; Dependence; Dissociation; Energy Transfer; Environment; Enzymes; Equilibrium; Eukaryotic Cell; Genes; Genetic; Genetic Transcription; Genome; Goals; Guanine; Guide RNA; Healthcare; Hematological Disease; High-Throughput Nucleotide Sequencing; Histones; Human; Immobilization; Incubated; Inherited; Kinetics; Knowledge; Label; Laboratories; Laboratory Research; Libraries; Malignant Neoplasms; Mammalian Cell; Manuscripts; Measures; Metabolic Diseases; Methods; Modification; Molecular; Molecular Biology; Molecular Conformation; Mus; Muscular Dystrophies; Mutation; Neurodegenerative Disorders; Nucleic Acids; Nucleosomes; Nucleotides; Outcome; Outcome Study; Pharmaceutical Preparations; Phase I Clinical Trials; Physiological; Plasmids; Play; Population; Process; Prokaryotic Cells; Proteins; RNA; RNA Folding; Reaction; Research Training; Resolution; Resources; Ribonucleoproteins; Role; Side; Specificity; Speed; Techniques; Technology; Testing; Therapeutic; Thermodynamics; Thymine; Time; Training; Universities; Variant; Work; Writing; base; biophysical analysis; biophysical techniques; design; endonuclease; experimental study; flexibility; fluorophore; gene function; genome editing; genomic locus; high throughput screening; human disease; improved; in vivo; laboratory experience; live cell imaging; millisecond; nanometer; next generation sequencing; nuclease; prediction algorithm; prevent; programs; single molecule; single-molecule FRET; skills; symposium; temporal measurement; tool; virtual

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.

项目概要/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 研究基因功能的一种方法是利用基因来消除其功能。 基因组编辑并研究动物模型中的规律间隔回文重复序列的结果。 (CRISPR) 相关蛋白，例如 Cas9，已成为首选的基因组编辑工具 Cas9 与其他方法相比易于使用，因此在医疗保健和生命科学应用中具有优势。 源自原核生物的核酸内切酶，被引导至基因组中的 20 个核苷酸序列，称为 Cas9 的原型间隔子可以通过改变引导 RNA 的序列来编程。 一种可编程的 DNA 切割蛋白，该技术已广泛应用于生命科学领域的基因研究。 功能，第一个基于 Cas9 的药物正在进入 1 期临床试验，但活性低下。 哺乳动物细胞是阻碍 Cas9 特异性增强变体和活性广泛使用的瓶颈。 我们和其他小组已经研究了如何优化和引导RNA。 尽管 Cas9 变体和不同的引导 RNA 会影响 DNA 结合、解旋和切割。 正确的 Cas9 复合物组装、Cas9 复合物结合和 DNA 的 Cas9 切割的重要性 染色质压缩哺乳动物细胞，我们对这些过程的分子细节的理解 在两个具体目标中，我建议通过首先量化和实际观察来填补这些知识空白。 Cas9 复合体如何组装及其核小体活性的时间目标之一是适应以前的情况。 开发了单分子测定法，我可以模拟共转录 RNA 折叠来研究分子 Cas9 与引导 RNA 折叠时的组装步骤，目标二将探究 Cas9 如何结合和裂解。 然后，我将使用高通量测序分析来研究 DNA 作为核小体周围 DNA 灵活性的函数。 量化结合动力学和平衡常数作为核小体周围 DNA 灵活性的函数。 这些实验的结果将极大地有助于我们对 CRISPR Cas 的基本理解 酶并将有助于开发基于 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、撰写手稿 总结我的发现并在会议上发言将增强量化和软技能。 实验室和约翰霍普金斯大学是这种研究培训的绝佳环境，主要是因为 获得广泛的专业知识和资源的机会。