Femto-seq: Targeted photo-biotinylation, pulldown and sequencing of locus and region-specific DNA from femtoliter volumes within individual cells

Femto-seq：从单个细胞内的飞升体积中对位点和区域特异性 DNA 进行靶向光生物素化、下拉和测序

• 批准号: 10587721
• 负责人: WARREN R ZIPFEL
• 金额: $ 42.13万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10587721
• 关键词: 3-Dimensional; Architecture; Biological; Biological Assay; Biotin; Biotinylation; Cell Line; Cell Nucleus; Cells; Cellular Stress; Chromatin; Collection; Computer software; Computers and Advanced Instrumentation; Consumption; Custom; Cytosol; DNA; DNA Sequence; DNA sequencing; Data; Dedications; Detection; Disease; Disease susceptibility; Enhancers; Ensure; Environment; Environmental Risk Factor; Eukaryota; Experimental Designs; Fluorescent in Situ Hybridization; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic Diseases; Genome; Genomic Segment; Genomics; Goals; Hand; Image; Image Analysis; In Situ; Individual; Knowledge; Label; Lasers; Libraries; Machine Learning; Manuals; Measures; Methods; Microfluidic Microchips; Microfluidics; Microscope; Microscopic; Microscopy; Molecular Conformation; Nuclear; Nuclear Structure; Optics; Physiologic pulse; Population; Population Analysis; Preparation; Procedures; Process; Protocols documentation; Regulation; Reporting; Research Personnel; Resolution; Role; Sampling; Scanning; Series; Shapes; Signal Transduction; Site; Stem Cell Research; Structure; Structure/Function Nuclei; System; Technology; Time; Transgenes; Treatment Protocols; Tube; Validation; cellular imaging; chromosome conformation capture; crosslink; design; design and construction; experimental study; flexibility; gene function; genome-wide; genomic locus; genomic tools; image processing; imaging modality; improved; insight; instrument; interest; multi-photon; new technology; novel; promoter; real-time images; response; sample fixation; software development; technology research and development; three dimensional structure; two-photon

Changes in the spatial organization of the genome are directly involved in gene regulation during differentiation, cellular stress responses and disease initiation. Existing approaches such as chromosome conformation capture (3C) methods and DNA fluorescent in situ hybridization (DNA-FISH) have provided information on the overall 3D structure of the nucleus at the chromatin level, providing critical insights into how our genomes are regulated. Nonetheless, new technologies are needed to uncover the finer details on the role of nuclear architecture, topological domains, and genomic interactions. Studies of gene regulation would benefit from a technology that fills the niche between the ensemble averaged 3C methods and the single cell, low throughput DNA-FISH approach. We have recently developed a novel optical technology we call “Femto-seq” that does just this – it allows users to obtain DNA sequence information from targeted femtoliter volumes within the nucleus of selected cells. Femto-seq provides a new way to examine genomic contacts near a specific gene locus or any nuclear region of interest (e.g. nuclear bodies). Using 3D localized two-photon excitation, we can photochemically biotinylate any region of the nucleus we can fluorescently label and identify in volumes which can be as small as a ½ of a femtoliter. The process is carried out on a population of cells using a combined two-photon/confocal microscope which images, locates fluorescently labeled regions of interest and then irradiates those regions using 700 nm femtosecond pulses to biotinylate the chromatin by photochemically cross-linking the DNA with a psoralen-biotin compound. Nuclei are isolated and the biotinylated DNA from the targeted region pulled down and sequenced. Because the cells are imaged to locate the regions of interest, they can also be screened for other parameters, allowing for the collection of targeted biotinylated DNA only from user selected cells within the cell population, providing single-cell like genomic information from a sub-set of cells within the population. We have proof-of-concept data from a cell line with a fluorescently labeled transgene we used as our targeted region, and show that we can obtain DNA highly enriched in transgene locus sequences. The goals of this Focused Technology Research and Development R01 project are to (Aim 1) design and construct a dual confocal/two- photon microscope capable of targeting and irradiating user selected regions-of-interest in a population of cells in a high-throughput automated fashion, (Aim 2) create a chromatin isolation pipeline based on novel microfluidic designs to efficiently purify and prepare the DNA for sequencing, and (Aim 3) demonstrate the improvement obtained from aims 1 and 2 in a series of Femto-seq experiments designed to produce quantitative metrics of improvement and to uncover new knowledge on how environmental signals may be relayed through the cytosol and into the nucleus. Femto-seq is a unique new way of investigating the spatial and regulatory relationships between DNA sequences and any microscopically visible region-of-interest in the nucleus.

基因组空间组织的变化直接参与分化过程中的基因调控，