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

 描述（由申请人提供）：我们提出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力学：顺序FISH和CRISPR成像。Cai实验室开发的seqFISH方法允许使用与条形码转录物和基因组基因座的连续多轮杂交在单细胞中检测多种核酸种类。Cai实验室将把这种方法扩展到DNA FISH，标记单细胞中的100个不同位点，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，Qi实验室开发了CRISPR成像工具，该工具使用标记有GFP的催化死亡Cas9，可以通过gRNA靶向特定的染色体基因座。我们将讨论实际的实现和潜在陷阱的解决方案。 我们将研究有多少异质性存在于单细胞中的染色体结构，以及它们如何与单个细胞的转录和表观遗传状态的功能措施相关。 小鼠胚胎干细胞是检测染色体结构异质性的天然系统，因为已经广泛研究了mESC的转录水平异质性。因此，它提供了最好的平台来检查染色体组织和转录调控之间的关系。 此外，seqFISH成像允许我们独立验证Hi-C结果。我们可以检查是否拓扑相关的结构域和染色体领域中观察到FISH，因为他们在HiC实验。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，并生成前所未有的染色体直接图像。 该项目中开发的方法可以很容易地传播，以供普遍使用。FISH已广泛用于染色体研究。seqFISH开发的协议可以很容易地通过任何实验室与原位杂交和显微镜的经验。该项目中开发的所有协议都将公开提供。

 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.