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， 环境和细胞压力，导致各种DNA损伤和基因 突变。许多蛋白质已经进化到可以快速检测、发送信号和修复DNA 活细胞内的损伤，形成一个被称为DNA的精心策划的网络 损伤响应（DDR）。不出所料，DDR缺陷，例如DNA修复蛋白 突变，往往与人类疾病，包括发育 异常，加速老化和常见癌症。过去几十年 生物化学和遗传学研究产生了丰富的知识 关于DDR因子的身份，它们在基因组维护中的作用，以及 当它们出错时是如何导致疾病的。然而，详细 DDR因子介导DNA修复时空参数仍然存在 很难捉摸DDR因素搜索并绑定到损坏的时间尺度是什么 活细胞中的DNA？解除武装、复员和重返社会因素是否形成了促进 准确修复？DNA损伤如何调节其他核DNA活动， 例如转录？该研究计划旨在解决这些基本问题。 通过研究DNA双链断裂（DSB）过程中的DDR动力学问题 修复. DSB是最常见的遗传毒性DNA损伤类型之一 在我们的身体里。最近，我们建立了一个实验平台， DDR因子的定量可视化和特定条件下的按需DSB诱导 基因组位点，并与第二规模的时间分辨率，一种能力，实现 通过将高分辨率荧光显微镜与超快的 （vf）我们实验室开创的CRISPR技术。在这里，我们将充分利用 这一新的平台，并全面映射DSB诱导的DDR动力学 因子，染色体易位，以及转录和cGAS的活性， 单个人体细胞这项研究将有力地补充DSB修复研究 传统上在试管中进行，并在合奏水平，提供 以前所未有的分辨率对DSB修复进行有价值的机械见解。