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

> **NIH NIH R21** · STANFORD UNIVERSITY · 2023 · $420,379

## Abstract

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.

## Key facts

- **NIH application ID:** 10725002
- **Project number:** 1R21HG013133-01
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Lei Stanley Qi
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $420,379
- **Award type:** 1
- **Project period:** 2023-08-01 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10725002

## Citation

> US National Institutes of Health, RePORTER application 10725002, Development of multi-color 3D super-localization LiveFISH and LiveFISH PAINT to investigate the chromatin dynamics at any genomic scale (1R21HG013133-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10725002. Licensed CC0.

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