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.