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 基因组的层次结构和动态决定了正确的 执行细胞类型特异性基因表达，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（例如多重检测） DNA FISH 已被开发用于表征 3D 基因组组织以及其扰动如何 然而，这些方法只能捕获疾病。 此外，染色质在固定时间点的构象和动态信息也丢失了。 位点标记方法需要繁琐的工作来创建细胞系，不能应用于原代细胞，并且不能 可以放大以追踪任何基因组长度尺度的基因组区域，因此，许多重要的生物学研究。 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基于 CRISPR（Clustered Regularly Interspaced Short Palindromic Repeats）的 成像技术 LiveFISH，可提供体外组装的荧光核糖核蛋白 (fRNP) 含有荧光团标记的引导 RNA 和 dCas9，可平铺活细胞中的基因组区域，而 LiveFISH 则为。 它在原代细胞中的 DNA 动态成像方面功能强大，但仅限于跟踪重复的基因组区域，这 该提案的主要目标是开发通用的基于成像的平台，称为 3D。 SL-LiveFISH 和 LiveFISH PAINT，用于跟踪任何基因组位点（重复或非重复）的动态 在任何基因组长度尺度上。 具体来说，我们将扩展之前的 LiveFISH 方法以针对任何基因组区域（包括重复的区域） 和非重复区域）在多种细胞类型中，包括原代细胞，在 3D 中具有高定位精度 （目标 1）此外，我们将开发 LiveFISH PAINT 来跟踪不同基因组长度的基因组区域。 规模并跟踪整个 Chr21 的动态（目标 2）将提供该项目的成功完成。 使用单活细胞成像研究 3D 基因组和基因之间因果关系的集成平台 它还将提供第一个关于整个染色体动力学的动态图景。 我们的工作意义重大，因为它将增进我们对不同基因组长度尺度的理解。 控制基因组在短期和长期时间尺度上的结构-功能关系的原则，并将 对许多实验室广泛有用。