Hi-resolution dynamic imaging of chromosomes in single cells by combined CRISPR imaging and sequential FISH

结合 CRISPR 成像和连续 FISH 对单细胞染色体进行高分辨率动态成像

• 批准号: 9003588
• 负责人: Long Cai
• 金额: $ 47万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9003588
• 关键词: Adopted; Back; Biological Assay; Bypass; Cell Culture Techniques; Cell Cycle Progression; Cells; Chromosome Structures; Chromosome Territory; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Color; DNA; DNA Methylation; Data; Data Set; Detection; Engineering; Epigenetic Process; Genes; Genetic Transcription; Genomics; Goals; Guide RNA; Heterogeneity; Image; Imaging Device; Immunofluorescence Immunologic; In Situ Hybridization; Individual; Label; Lead; Length; Life; Mammalian Cell; Measurement; Measures; Methods; Microscopy; Mus; Nucleic Acids; Play; Positioning Attribute; Proteins; Protocols documentation; Qi; RNA; RNA-Binding Proteins; Research Personnel; Resolution; Role; Solutions; System; Technology; Time; Tissues; Transcript; Transcriptional Regulation; Translating; Validation; Work; cell fixing; embryonic stem cell; experience; graduate student; imaging modality; movie; public health relevance; reconstruction; research study; single cell analysis; temporal measurement; tool

 DESCRIPTION (provided by applicant): We propose to visualizing chromosome structure and dynamics in single cells by a combination of two approaches: sequential FISH and CRISPR imaging. seqFISH methods developed in the Cai lab allows multiple nucleic acid species to be detected in single cells using sequential rounds of hybridization to barcode transcripts and genomic loci. The Cai lab will extend this method to DNA FISH to tag 100 different loci in single cells, providing a snapshot of the structure of the chromosome in individual cells. To image the dynamic of these loci, the Qi lab has developed CRISPR imaging tools which uses a catalytically dead Cas9 labeled with GFP that can be targeted to particular chromosomal loci by gRNAs. We will discuss the practical implementations and solutions to potential pitfalls. We will investigate how much heterogeneity exist in chromosome structures in single cells and how they are correlated to functional measures of transcriptional and epigenetic states of individual cells. Mouse embryonic stem cells are a natural system to examine heterogeneity in chromosome structure, because mESCs have been extensively studied for transcriptional level heterogeneity. Thus, it offers the best platform to examine the relationship between chromosomal organization and transcriptional regulation. In addition, seqFISH imaging allows us to validate the Hi-C results independently. We can examine whether topological associated domains and chromosomal territories are observed in FISH as they have in HiC experiments. We will characterize the distribution of the heterogeneities in the chromosome structures at various length scales in single cells and generate unprecedented direct images of the chromosomes. The methods developed in this project can be easily disseminated for general use. FISH has been used extensively for chromosome studies. Developed protocols for seqFISH can be readily adopted by any lab with experience with in situ hybridization and microscopy. All protocols developed in the project will be made available publicly.

 描述（由申请人提供）：我们建议通过结合两种方法来可视化单细胞中的染色体结构和动态：蔡实验室开发的连续 FISH 和 seqFISH 方法允许使用单细胞中检测多种核酸。 Cai 实验室将这种方法扩展到 DNA FISH，以标记单个细胞中的 100 个不同基因座，从而提供单个细胞中染色体结构的快照。为了对这些位点的动态进行成像，Qi 实验室开发了 CRISPR 成像工具，该工具使用 GFP 标记的催化死亡 Cas9，可以通过 gRNA 靶向特定的染色体位点。我们将讨论潜在缺陷的实际实施和解决方案。单细胞染色体结构存在很大的异质性，以及它们如何与单个细胞的转录和表观遗传状态的功能测量相关。 小鼠胚胎干细胞是检查染色体结构异质性的天然系统，因为 mESC 已针对转录水平异质性进行了研究，因此，它提供了广泛检查染色体组织和转录调控之间关系的最佳平台。我们可以独立验证 Hi-C 结果。我们可以检查 FISH 中是否观察到拓扑相关结构域和染色体区域，我们将描述异质性的分布。该项目开发的方法可以轻松传播以用于染色体研究。任何具有原位杂交和显微镜经验的实验室都将公开该项目中开发的所有协议。