Developments of MERFISH for functional genomics and spatial epigenomics

Abstract

The recent development of spatial omics methods, enables spatial profiling of transcriptome and 3D structure of genomes at a single-cell level. Expanding the repertoire of spatial omics tools, a spatial epigenomics method will accelerate our understanding of the spatial regulation of cell and tissue functions. Furthermore, an imaging-based pooled-library CRISPR screening approach, potentially compatible with spatial omics phenotype, can facilitate the discovery of the underlying genetic factors regulating the spatial characteristics of multi-omics. The first part of this thesis describes my efforts to develop a method for spatially resolved profiling of epigenomes in single cells using in-situ tagmentation and transcription followed by highly multiplexed imaging. We profiled two histone modifications, H3K4me3 and H3K27ac, canonical markers for active promoters and enhancers, providing high-throughput and high-resolution measurements of spatial distributions of active promoters and putative enhancers in embryonic and adult mouse brains. Our results further revealed putative enhancer hubs regulating the expression of developmentally important genes. The second part of this thesis reports my efforts to develop an all imaging-based pooled-library CRISPR screening approach that combines high-content phenotype imaging with high-throughput single guide RNA (sgRNA) identification in individual cells. In this approach, sgRNAs are codelivered to cells with corresponding barcodes using a lentiviral delivery system. Multiplexed error-robust fluorescent in situ hybridization (MERFISH) is used to read out the barcodes and hence identify the sgRNAs with high accuracy. We used this approach to screen 162 sgRNAs targeting 54 RNA-binding proteins for their effects on RNA localization to nuclear compartments and uncovered previously unknown regulatory factors for nuclear RNA localization. We revealed both positive and negative regulators for the nuclear speckle localization of a long noncoding RNA, MALAT1, suggesting a dynamic regulation of lncRNA localization in subcellular compartments. With the approaches of spatial profiling of the transcriptome, 3D genome, and epigenome and pooled-library CRISPR screen on MERFISH phenotypes, we envision that those developments will provide insight into how multi-omics are coordinated in single cells, advancing our understanding of how gene expression is spatiotemporally regulated during cellular processes.