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

摘要 哺乳动物细胞中3D基因组的层次组织和动力学决定了合适的基因组结构。 细胞类型特异性基因表达的执行，并与细胞功能和人类疾病密切相关。 多种基于测序的方法（例如Hi-C）和基于成像的方法（例如多路复用的方法）可以用于在细胞中进行测序。 DNA FISH已被开发用于表征3D基因组组织以及其 组织影响发育并导致疾病。然而，这些方法只能捕获 在固定时间点染色质构象和动态信息丢失。此外，许多以前的DNA 基因座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 可以按比例放大以跟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 关于染色质组织，动力学和基因之间的功能关系的问题 转录仍然是难以捉摸的。 我们的实验室最近开发了一种基于CRISPR（重复的规则间隔短回文重复序列）的 成像技术，LiveFISH，其提供体外组装的荧光核糖核蛋白（fRNP） 包含荧光团标记的引导RNA和dCas 9以平铺活细胞中的基因组区域。虽然LiveFish是 它在原代细胞中的DNA动力学成像方面很强大，但仅限于跟踪重复的基因组区域， 大大限制了它的使用。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，我们将扩展以前的LiveFISH方法，以靶向任何基因组区域（包括重复序列）。 和非重复区域）在包括原代细胞在内的多种细胞类型中以3D的高定位精度 (Aim 1）。此外，我们将开发LiveFISH PAINT，以跟踪不同基因组长度的基因组区域 尺度和跟踪整个Chr 21的动态（目标2）。该项目的成功完成将提供 使用单个活细胞成像的集成平台，研究3D基因组和基因之间的因果关系 在不同细胞类型中的调节。它还将提供第一个动态图片的整个染色体动态， 不同基因组长度的活细胞。我们的工作意义重大，因为它将促进我们对 在短期和长期时间尺度上控制基因组结构-功能关系的原则， 对许多实验室都有很大的帮助。

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.