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， 细胞应激反应和疾病引发。现有的方法，如染色体构象捕获 (3C)方法和DNA荧光原位杂交（DNA-FISH）提供了关于整体3D 在染色质水平上研究细胞核的结构，为我们的基因组如何调控提供重要的见解。 尽管如此，需要新技术来揭示核结构作用的更精细细节， 拓扑结构域和基因组相互作用。基因调控的研究将受益于一种技术， 填补了整体平均3C方法和单细胞低通量DNA-FISH之间的空白 approach.我们最近开发了一种新的光学技术，我们称之为"Femto-seq"，它 允许用户从选定的细胞核内的靶向毫微微体积中获得DNA序列信息， 细胞Femto-seq提供了一种新的方法来检查特定基因位点或任何核基因附近的基因组接触 感兴趣的区域（例如核体）。使用3D局域双光子激发，我们可以光化学 生物素化细胞核的任何区域，我们可以荧光标记和识别的体积可以小到 半个飞升。该过程使用组合的双光子/共聚焦激光器在细胞群上进行。 显微镜，其成像、定位感兴趣的荧光标记区域，然后照射这些区域 使用700 nm飞秒脉冲，通过光化学交联DNA与 紫杉醇-生物素化合物。分离细胞核，并将来自靶区域的生物素化DNA拉下 和序列测定因为细胞被成像以定位感兴趣的区域，所以它们也可以被筛选， 其他参数，允许仅从用户选择的细胞中收集靶向生物素化DNA， 细胞群体，提供来自群体内细胞子集的单细胞样基因组信息。我们 有来自一个细胞系的概念验证数据，我们用荧光标记的转基因作为我们的目标区域， 并且表明我们可以获得在转基因位点序列中高度富集的DNA。本专题的目标 技术研究和开发R01项目是（目标1）设计和建造一个双共焦/双- 能够瞄准和照射用户在细胞群中选择的感兴趣区域的光子显微镜 以高通量自动化方式，（目标2）创建基于新型微流体的染色质分离管道， 设计，以有效地纯化和制备用于测序的DNA，并（目的3）证明改进 在一系列Femto-seq实验中从目标1和2获得，这些实验旨在产生 改善和发现新的知识，环境信号如何可能通过细胞质中继 并进入细胞核。Femto-seq是一种独特的研究空间和调控关系的新方法 DNA序列和细胞核中任何显微镜可见的感兴趣区域之间的关系。