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

人类基因组的完整性不断受到挑战 环境和细胞应激，导致各种DNA损伤和基因 突变。许多蛋白质已经进化成能够快速检测、发送信号和修复DNA 破坏活细胞内部，形成一个被称为DNA的有序网络 损害反应(DDR)。不出所料，DDR缺陷，如DNA修复蛋白 突变，通常与人类疾病有关，包括发育 畸形、加速衰老和常见癌症。过去几十年来， 生化和基因研究产生了丰富的知识 关于DDR因子的特性，它们在基因组维持中的作用，以及 当他们走错路时，他们是如何促成疾病的。然而，详细的 DDR因子介导DNA修复的时空参数仍然存在 在很大程度上是难以捉摸的。DDR因子搜索并绑定损坏的时间尺度是什么 活细胞中的DNA？DDR因素是否形成特定的结构以促进 精准修复？DNA损伤如何调控其他核DNA活动， 比如抄写？这项研究计划旨在解决这些基本问题 DNA双链断裂(DSB)过程中DDR动力学研究中的问题 修理。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，允许 DDR因素的定量可视化和特定情况下的按需DSB诱导 基因组位置和第二尺度的时间分辨率，实现了 通过将高分辨率荧光显微镜与非常快速的 (VF)本实验室首创的CRISPR技术。在这里，我们将充分利用 这一新的平台并全面映射了DSB诱导的DDR动力学 因子、染色体易位以及转录和cGAS活性 单个人类细胞。这项研究将有力地补充DSB修复研究 常规地在试管和合奏水平上表演，提供 以前所未有的决心对DSB修复提供有价值的机械性见解。

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.