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RNA, which serves as a pivotal intermediary between DNA and proteins, plays a crucial role in shaping cellular functions and morphology. Generations of different techniques have been developed to enhance the understanding of RNA with respect to its sequence, function and spatial localization. While recent advancements have produced RNA detection methods capable of providing high-resolution insights into various RNA species, the disassociation process of these methods sacrifices the spatial arrangement of cells and native cell morphology. This calls for the need of in situ detection of RNA in its natural states, which would retain the constitution of cells. Multiplexed in situ RNA detection methods have evolved rapidly in the past decade. These techniques enable optical profiling and decoding of RNA, allowing for the acquisition of both transcriptomic and spatial features of the cells. This thesis focuses on the study of two in situ RNA detection methods: RNAmap and CRISPRmap. These approaches significantly increase signal level, and the cyclical readout probe hybridization scheme enables multiplexed readout, with the additional benefit of not being dependent on third-party sequencing reagents. The first chapter of this thesis sets forth an overview of different RNA detection methods, with an emphasis on RNAmap and CRISPRmap. The second chapter details the optimization of RNAmap in a novel organism, Oxytricha trifallax. RNAmap is able to detect different mRNA species in Oxytricha with robust signal intensity and specificity. Fluorescent in situ hybridization detection of telomeres in Oxytricha cells at different developmental time points also unveil a series of genome arrangement activities and nuclear morphology changes, which introduces novel biological questions for future research. The third chapter outlines the application of CRISPRmap, a pooled optical screening assay developed in our lab, to screen for novel modulators of WRN protein, an interesting synthetic lethal target of microsatellite unstable cancers. A pilot screen perturbed known modulators, and demonstrated expected effects on WRN’s function. After testing out CRISPRmap’s efficacy, we ventured from perturbing known modulators and expanded our screen to the entire kinome, exploring kinase regulation of WRN’s diverse function in DNA damage repair and cell viability. |
