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.

摘要