Mapping the Cellular Responses to DNA Double-Strand Breaks Using On-Demand CRISPR technologies and High-resolution Fluorescence Microscopy

使用按需 CRISPR 技术和高分辨率荧光显微镜绘制细胞对 DNA 双链断裂的反应

• 批准号: 10715720
• 负责人: Yang Liu
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10715720
• 关键词: Acceleration; Address; Aging; Binding; Biochemical; CRISPR/Cas technology; Cells; Cellular Stress; Chromatin; Chromosomal translocation; Complement; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Gene; Defect; Development; Disease; Double Strand Break Repair; Fluorescence Microscopy; Gene Mutation; Genetic Research; Genetic Transcription; Genome; Human; Human Genome; Immune system; Innate Immune System; Kinetics; Knowledge; Link; Maintenance; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Mutation; Nuclear; Pathway interactions; Proteins; Research; Research Proposals; Resolution; Role; Signal Transduction; Structure; Testing; Tube; Visualization; biophysical techniques; environmental stressor; genetic approach; genome integrity; genomic locus; genotoxicity; human disease; insight; novel; programs; rapid detection; repaired; response; spatiotemporal; temporal measurement

The integrity of the human genome is constantly challenged by environmental and cellular stresses, resulting in various DNA damage and gene mutations. Many proteins have evolved to rapidly detect, signal, and repair DNA damage inside living cells, forming an orchestrated network known as DNA damage response (DDR). Unsurprisingly, DDR defects, such as DNA repair protein mutations, are often linked to human diseases, including developmental abnormalities, accelerated aging, and common cancers. The past decades of biochemical and genetic research have generated a wealth of knowledge regarding the identities of DDR factors, their roles in genome maintenance, and how they contribute to the diseases when they go awry. However, the detailed spatiotemporal parameters by which DDR factors mediate DNA repair remain largely elusive. What timescales do DDR factors search for and bind to damaged DNA in living cells? Do DDR factors form specific structures to facilitate an accurate repair? How does DNA damage regulate other nuclear DNA activities, such as transcription? This research program aims to address these fundamental questions by investigating DDR dynamics during DNA double-strand break (DSB) repair. DSB is one of the most genotoxic DNA damage types frequently occurring in our bodies. Recently, we have established an experimental platform that allows quantitative visualization of DDR factors and on-demand DSB induction at specific genomic loci and with a second-scale temporal resolution, a capability achieved by marrying high-resolution fluorescence microscopy with the very fast (vf)CRISPR technique pioneered by our lab. Here, we will take full advantage of this novel platform and comprehensively map the DSB-induced dynamics of DDR factors, chromosome translocation, and activities of transcription and cGAS in single human cells. This study will strongly complement DSB repair research conventionally performed in test tubes and at the ensemble level, providing valuable mechanistic insights into DSB repair with unprecedented resolutions.

人类基因组的完整性不断受到挑战 环境和细胞应力，导致各种DNA损伤和基因 突变。许多蛋白质已经演变为快速检测，信号和修复DNA 活细胞内部的损坏，形成一个精心策划的网络，称为DNA 伤害响应（DDR）。毫不奇怪，DDR缺陷，例如DNA修复蛋白 突变通常与人类疾病有关，包括发展 异常，加速衰老和常见癌症。过去几十年 生化和遗传研究产生了丰富的知识 关于DDR因素的身份，它们在基因组维持中的作用以及 当他们出现问题时，它们如何促进疾病。但是，详细 DDR因子介导DNA修复的时空参数仍在 在很大程度上难以捉摸。 ddr因素搜索并结合损坏 活细胞中的DNA？ DDR因素形成特定结构以促进 准确维修？ DNA损伤如何调节其他核DNA活性， 例如转录？该研究计划旨在解决这些基本 通过研究DNA双链断裂期间的DDR动力学（DSB）来提出问题 维修。 DSB是经常发生的最遗传毒性DNA损伤类型之一 在我们的体内。最近，我们建立了一个实验平台，允许 DDR因子和点播DSB诱导的定量可视化在特定 基因组基因局和第二尺度的时间分辨率，达到了能力 通过将高分辨率荧光显微镜与非常快的 （VF）CRISPR技术由我们的实验室开创。在这里，我们将充分利用 这个新颖的平台，并全面绘制DSB诱导的DDR动力学 因素，染色体易位以及转录和CGA的活动 单个人类细胞。这项研究将强烈补充DSB维修研究 传统上在测试管和集合级别上执行 通过前所未有的决议对DSB维修的有价值的机械洞察力。