Development of multi-color 3D super-localization LiveFISH and LiveFISH PAINT to investigate the chromatin dynamics at any genomic scale

开发多色 3D 超定位 LiveFISH 和 LiveFISH PAINT，以研究任何基因组规模的染色质动态

• 批准号: 10725002
• 负责人: Lei Stanley Qi
• 金额: $ 42.04万
• 依托单位: STANFORD UNIVERSITY
• 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 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10725002
• 关键词: 3-Dimensional; Adopted; Affect; Architecture; Behavior; Benchmarking; Biological; Cell Line; Cell Nucleus; Cell physiology; Cells; Chemistry; Chromatin; Chromosome Structures; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Color; DNA; Data; Defect; Development; Disease; Dyes; Electroporation; Enhancers; Ensure; Epigenetic Process; Etiology; Fluorescent Dyes; Fluorescent in Situ Hybridization; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomic DNA; Genomic Segment; Genomics; Goals; Guide RNA; Hi-C; Hour; Human Genome; Image; Imaging technology; In Vitro; Knock-in; Label; Length; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Methods; Molecular Conformation; Monitor; Neurodevelopmental Disorder; Neurons; Noise; Oligonucleotides; Outcome; Performance; Reagent; Research Personnel; Resolution; Ribonucleoproteins; Signal Transduction; Structure; Structure-Activity Relationship; T-Lymphocyte; Techniques; Technology; Time; Transfection; Work; automated algorithm; biophysical model; cancer cell; cell type; chromosome conformation capture; design; fluorophore; genomic locus; human disease; imaging approach; induced pluripotent stem cell; live cell imaging; mammalian genome; nanoparticle; neuropsychiatric disorder; new technology; novel strategies; promoter; scale up; stem; technology platform; tool; whole genome

ABSTRACT The hierarchical organization and dynamics of the 3D genome in mammalian cells determines the proper execution of cell type-specific gene expression and is closely related with cellular function and human disease. A variety of sequencing-based approaches such as Hi-C and imaging-based approaches such as multiplexed DNA FISH have been developed to characterize 3D genome organization and how perturbations in its organization affect development and cause diseases. However, these approaches can only capture the conformation of chromatin at a fixed time point and dynamic information is lost. In addition, many previous DNA locus labelling methods require tedious effort to create cell lines, cannot be applied to primary cells, and cannot be scaled up to track genomic regions of any genomic length scales. As a result, many significant biological questions regarding the functional relationship between chromatin organization, dynamics, and gene transcription still remains elusive. Our lab has recently developed a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based imaging technology, LiveFISH, which delivers in vitro assembled fluorescent ribonucleoproteins (fRNPs) containing fluorophore-labelled guide RNAs and dCas9 to tile a genomic region in live cells. While LiveFISH is powerful in imaging DNA dynamics in primary cells, it is limited to tracking repetitive genomic regions, which greatly limits its use. The major goal of this proposal is to develop versatile imaging-based platforms, termed 3D SL-LiveFISH and LiveFISH PAINT, to track the dynamics of any genomic locus (repetitive or non-repetitive) and on any genomic length scale. Specifically, we will expand the previous LiveFISH approach to target any genomic region (including repetitive and non-repetitive regions) in a variety of cell types including primary cell with high localization precision in 3D (Aim 1). Furthermore, we will develop LiveFISH PAINT to track genomic regions at different genomic length scales and to track the dynamics of the whole of Chr21 (Aim 2). Successful completion of the project will provide an integrated platform using single live cell imaging to study the causality between the 3D genome and gene regulation in diverse cell types. It will also provide the first dynamic picture of whole chromosome dynamics in live cells at different genomic length scales. Our work is significant because it will advance our understanding of the principles governing the genome’s structure-function relationship across short and long time scales and will be broadly useful for many labs.

抽象的 哺乳动物细胞中3D基因组的分层组织和动力学决定了适当的 细胞类型特异性基因表达的执行，与细胞功能和人类疾病密切相关。 各种基于测序的方法，例如HI-C和基于成像的方法，例如多路复用 已经开发出DNA鱼来表征3D基因组组织以及如何在其中扰动 组织影响发展并引起疾病。但是，这些方法只能捕获 染色质在固定时间点的构象，动态信息丢失。另外，许多以前的DNA 基因座标记方法需要繁琐的努力来创建细胞系，不能应用于主单元，也不能 缩放以跟踪任何基因组长度尺度的基因组区域。结果，许多重要的生物学 有关染色质组织，动力学和基因之间功能关系的问题 转录仍然难以捉摸。 我们的实验室最近开发了一个CRISPR（群集定期间隔短的短质重复序列）基于 成像技术，LiveFish，它提供体外组装的荧光核糖核蛋白（FRNP） 含有荧光团标记的指南RNA和DCAS9，以在活细胞中的基因组区域瓷砖。虽然livefish是 在原代细胞中成像DNA动力学的功能强大，它仅限于跟踪重复的基因组区域，该区域 极大地限制了其使用。该提案的主要目标是开发基于多功能成像的平台，称为3D SL-Live鱼和Livefish油漆，以跟踪任何基因组基因座（重复或不重视）的动态 在任何基因组长度尺度上。 具体而言，我们将扩展以前的LiveFish方法以靶向任何基因组区域（包括重复性 在多种细胞类型中，包括在3D中具有较高定位精度的主要细胞中的非重复区域） （目标1）。此外，我们将开发LiveFish油漆以跟踪不同基因组长度的基因组区域 鳞片并跟踪CHR21的整个动力学（AIM 2）。该项目的成功完成将提供 使用单个活细胞成像研究3D基因组和基因之间的因果关系的集成平台 潜水细胞类型的调节。它还将提供整个染色体动力学的第一个动态图片 不同基因组长度尺度的活细胞。我们的工作很重要，因为它将提高我们对 在短期和长时间范围内管理基因组结构功能关系的原则，将会 对于许多实验室而言，广泛有用。