Development of Single-Cell SPRITE: a Tool for Measuring Heterogeneity of 3D DNA Organization

Citation

Arrastia, Mary Villanueva (2022) Development of Single-Cell SPRITE: a Tool for Measuring Heterogeneity of 3D DNA Organization. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/w70x-2294. https://resolver.caltech.edu/CaltechTHESIS:10012021-223453672

Abstract

Across eukaryotic cells, DNA from each nucleus is organized in three dimensions in order to help regulate transcriptional activity. Decades of chromosome capture technologies have revealed fundamental chromatin structures, providing information about how DNA is assembled genome-wide. The majority of these methods utilize direct physical ligation of DNA molecules to generate pairwise interactions, which have provided information about short-range interactions and intra-chromosomal structures. Recent technologies have moved toward identifying multiple DNA interactions simultaneously without physical ligation of DNA molecules, revealing information about long-range interactions and inter-chromosomal structures. One of the biggest limitations of these methods is that they only study DNA organization in bulk, which misses the heterogeneity of chromosomal structures at the single-cell level. As a result, single-cell chromosome capture methods have been developed to begin probing into the cell-to-cell variability of DNA organization and answer long-standing questions regarding single-cell structure. However, single-cell methods are currently limited to identifying low-resolution, intra-chromosomal DNA interactions with few numbers of cells. This creates a need for an improved, high-throughput single-cell method that can capture high-resolution structures and simultaneous mapping of both intra- and inter-chromosomal interactions to better elucidate single-cell DNA organization. In this thesis, we describe the development of 'single-cell split-pool recognition of interactions by tag extension' (scSPRITE), a single-cell chromosome capture method that allows for mapping of high-resolution, intra- and inter-chromosomal structures across thousands of cells. Through scSPRITE, we were not only able to reveal fundamental information about single-cell DNA organizations, but we can also quantitatively measure the variability of DNA interactions from cell to cell.

Thesis Files