Using and Adapting the Functional Genomic Toolkit for Exploration of Environmental Stress Responses

Abstract

All organisms maintain homeostasis in the face of environmental flux through conserved and lineage-specific stress response mechanisms. However, our ability to dissect these mechanisms at a molecular level remains confined to a small number of model organisms. The functional genomic tools that have transformed mammalian biology — CRISPR-based screening, RNA interference, and scalable nucleic acid delivery — have not been meaningfully extended to non-model taxa. The lack of well-developed functional genomic tools in those spaces is an impediment to many lines of inquiry. At the same time, even in well-established mammalian models, a gap between experimental paradigms and physiological reality exists: laboratory studies of environmental toxicant stress largely employ acute, high-concentration exposures that do not closely resemble the chronic, low-concentration paradigm characteristic of most real-world environmental toxicant exposure in humans. This dissertation makes progress in both areas — the technological gap limiting functional genomics in non-model marine invertebrates, and the conceptual gap between acute and chronic stress biology — through work in the sea urchin Lytechinus variegatus and in human cell culture.In the first chapter, I characterize an embryonic cell line derived from L. variegatus blastulae using integrated single-cell RNA sequencing. By comparing cultured cells to a developmental transcriptomic atlas, I establish their identity, evaluate what they retain of their developmental origin, and helping to establish their suitability as an in vitro platform for functional studies. In the second chapter, I develop a suite of genetic perturbation tools for the sea urchin. I design and test expression cassettes and lentiviral vectors for transgene delivery in urchin cell lines, demonstrate for the first time that RNA interference is viable in the sea urchin embryo — overturning a long-standing assumption in the field — and show that lipid nanoparticles (LNPs) can deliver nucleic acid cargo to live embryos. In the third chapter, I use genome-scale CRISPR knockout screens in human U2OS cells to compare the genetic requirements for survival under chronic, low-level arsenite exposure (4 μM, continuous) versus acute, high-intensity exposure. Arsenite is a pervasive groundwater contaminant estimated to affect over 230 million people globally, yet its study in the laboratory has been dominated by acute dosing paradigms. I map screen hits onto curated protein interaction databases and apply graph-based and spatial analysis frameworks to resolve functional modules within the ubiquitin proteasome system (UPS), complementing standard enrichment approaches that lack power or resolution within sub-genomic, functionally related gene sets. I validate key findings using flow cytometry and quantitative imaging. The sea urchin work establishes that L. variegatus blastula-derived cells retain identifiable transcriptomic signatures of their developmental origin and are amenable to lentiviral transduction. The demonstration of RNAi and LNP-mediated delivery in urchin embryos closes a thirty-year methodological gap and establishes the last necessary technological pillar — scalable nucleic acid delivery — on which high-throughput forward genetic screening in marine invertebrates can be built. The CRISPR screens in U2OS cells reveal that chronic and acute arsenite stress engage distinct functional modules within the ubiquitin network, with chronic exposure producing a pattern of genetic dependency that is not a subset of the acute response but a separable state with its own conditionally essential genes. These findings support the conclusion that the cellular logic of environmental stress response is regime-dependent, and that the UPS encodes distinct regulatory programs for different modes of the same stressor. Together, this work advances both the experimental infrastructure for functional genomics in non-model organisms and our mechanistic understanding of how cells distinguish and respond to different temporal patterns of environmental insult.