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; 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（通过应用程序提供）：我们建议通过两种方法的组合来可视化染色体的结构和动态：顺序鱼类和CRISPR成像。在CAI实验室开发的Seq鱼方法允许在单个细胞中使用顺序的杂交与条形码转录本和基因组基因座进行多种核酸物种。 CAI实验室将将这种方法扩展到DNA FISH，以标记单个细胞中的100个不同当地人，从而提供单个细胞中染色体结构的快照。为了想象这些当地人的动态，QI实验室开发了CRISPR成像工具，该工具使用了用GFP标记的催化死亡CAS9，该CAS9可以针对GRNA的特定染色体基因座。我们将讨论潜在陷阱的实际实施和解决方案。我们将研究单细胞中染色体结构中存在多少异质性，以及它们与单个细胞转录和表观遗传态的功能测量的相关性。 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已被广泛研究以进行转录水平的异质性。这是检查染色体组织与转录调节之间关系的最佳平台。此外，SeqFish成像使我们能够独立验证HI-C结果。我们可以检查我们是否将表征单个细胞中各个长度尺度的染色体结构中异质性的分布，并产生染色体前所未有的直接图像。该项目中开发的方法可以轻松传播供一般使用。鱼已被广泛用于染色体研究。任何具有原位杂交和显微镜的经验的实验室都可以轻松地采用开发的Seq鱼方案。项目中开发的所有协议将公开提供。