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基因组的分层组织和动力学决定了适当的 执行细胞类型特异的基因表达，与细胞功能和人类疾病密切相关。 各种基于测序的方法，如Hi-C和基于成像的方法，如多路传输 DNA FISH已被开发用于表征3D基因组组织以及其 组织影响发展，引发疾病。然而，这些方法只能捕获 染色质在固定时间点的构象和动态信息丢失。此外，许多以前的DNA 位置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，也不能 被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、动力学和基因之间功能关系的问题 抄写仍然难以捉摸。 我们实验室最近开发了一种基于CRISPR(聚类规则间隔短回文重复)的方法 成像技术，LiveFISH，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，以在活细胞中划分基因组区域。而LiveFISH是 在原代细胞中成像DNA动力学的强大功能，它仅限于跟踪重复的基因组区域，这 极大地限制了它的使用。这项提议的主要目标是开发基于成像的多功能平台，称为3D SL-LiveFISH和LiveFISH Paint，跟踪任何基因组基因座(重复或非重复)的动态，以及 在任何基因组长度尺度上。 具体地说，我们将把以前的LiveFISH方法扩展到针对任何基因组区域(包括重复的 和非重复区域)在各种细胞类型中，包括3D中具有高定位精度的初级细胞 (目标1)。此外，我们还将开发LiveFISH Paint来跟踪不同基因组长度的基因组区域 标度和跟踪整个克莱斯21号的动态(目标2)。该项目的成功完成将提供 利用单个活细胞成像研究3D基因组和基因之间因果关系的集成平台 对不同细胞类型的调控。它还将提供第一张关于整个染色体动力学的动态图片 不同基因组长度尺度下的活细胞。我们的工作意义重大，因为它将促进我们对 支配基因组在短时间和长时间尺度上的结构-功能关系的原理和将 对许多实验室都很有用。

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.