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)：我们建议通过两种方法的组合来可视化单细胞中的染色体结构和动态：顺序FISH和CRISPR成像。Cai实验室开发的SeqFISH方法允许使用条形码转录本和基因组位置的连续几轮杂交在单个细胞中检测到多种核酸物种。Cai实验室将把这种方法扩展到DNA FISH，以标记单细胞中的100个不同的基因座，提供单个细胞中染色体结构的快照。为了对这些基因座的动态进行成像，齐实验室开发了CRISPR成像工具，它使用了一个标记有GFP的催化死亡的Cas9，它可以通过gRNA靶向特定的染色体基因座。我们将讨论潜在陷阱的实际实现和解决方案。我们将调查单个细胞的染色体结构存在多大的异质性，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联。 小鼠胚胎干细胞是检测染色体结构异质性的自然系统，因为mESCs已被广泛研究转录水平的异质性。因此，它为研究染色体组织和转录调控之间的关系提供了最好的平台。此外，seqFISH成像允许我们独立地验证Hi-C结果。我们可以检查是否像在HIC实验中那样，在FISH中观察到了拓扑相关结构域和染色体区域。我们将描述单细胞中不同长度尺度的染色体结构中异质性的分布，并产生前所未有的直接染色体图像。在这个项目中开发的方法可以很容易地传播到一般用途。FISH已被广泛用于染色体研究。开发的seqFISH方案可以很容易地被任何有原位杂交和显微镜经验的实验室采用。在该项目中开发的所有协议都将公开提供。