Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular, Cellular, and Developmental Biology

First Advisor

Isaacs, Farren

Abstract

Cells are self-replicating information processing systems that maintain control of internal state by gathering and expending chemical energy. Programming these cellular states requires complementary technological paradigms: genome editing methods define the phase space of possible cellular behaviors by modifying an underlying genetic blueprint, and transcriptional and allosteric control systems enable precise navigation within that defined space through spatiotemporal and environmentally responsive regulation. In this sense, genome editing and gene regulation tools work synergistically to expand ability to perturb, control, and monitor cellular function. No single genome editing or control paradigm optimally addresses all applications: eukaryotic MAGE (eMAGE) excels at generating combinatorial diversity across populations, while nuclease-based systems paired with homology-directed repair leverage host-specific repair pathways, providing predictable and efficient targeted modifications at defined loci. Similarly, each control system paradigm offers unique advantages: transcriptional regulation provides gene-independent control, while allosteric systems enable host-independent portability. Advancing both genome editing and control system approaches expands our capacity to efficiently explore the parameter space of possible genomes and subsequently the phenotypic space for a given genome.Current genome editing technologies face significant limitations: oligonucleotide-mediated recombineering in eukaryotes suffers from low efficiency without mismatch repair suppression, nuclease multiplexing induces cytotoxicity and chromosomal rearrangements, and existing inducible systems lack the orthogonality and dynamic range needed for complex synthetic circuits. In this dissertation, I seek to address these constraints through genetic and protein engineering. I begin in Chapter 1 with a comprehensive background on genome editing technologies, DNA repair pathways, and synthetic transcriptional and allosteric control systems that provide background for the subsequent chapters. In Chapter 2, I sought to identify the molecular mechanism underlying eMAGE in S. cerevisiae, hypothesizing that Rad59-dependent DNA repair pathways facilitate oligonucleotide incorporation. Through systematic genetic knockouts and synthetic reconstruction attempts using RPA-SSB fusion proteins, I determined that salvage recombination, break-induced replication, and other canonical Rad59-dependent processes do not mediate eMAGE editing. These mechanistic insights establish critical constraints for future studies and highlight the need for novel approaches to achieve efficient oligonucleotide-mediated editing in eukaryotes comparable to bacterial recombineering systems. In Chapter 3, I aimed to overcome the efficiency limitations of eMAGE that restricted its utility for combinatorial genetics and applied these improvements to study nuclear hormone receptor dysfunction. By optimizing oligonucleotide design, developing dual-marker co-selection strategies, and engineering transient mismatch repair suppression, I achieved up to 90% editing efficiency in cell populations and extended the editing range to 20 kilobases with >40% efficiency. These advances enabled the generation of combinatorial mutations in nuclear hormone receptor ligand-binding domains, revealing previously unknown autoactivating variants that provide mechanistic insights into receptor-associated cancers and demonstrate the utility of multiplex editing for dissecting complex genetic interactions. In Chapter 4, I sought to expand the toolkit for programmable cellular control by engineering both orthogonal synthetic transcription factors and allosteric CRISPR systems responsive to small molecules. Through systematic domain swapping, I created four mutually orthogonal hormone-responsive transcription factors, then extended this principle to engineer Cas9 variants with superior on/off switching controlled by non-physiological ligands. Most significantly, I developed the first functional allosteric dCas9 in eukaryotes, providing a tool for reversible, titratable gene regulation without permanent genome modification that will enable new therapeutic strategies and temporal control of gene expression. In Chapter 5 I propose future experiments leveraging phase separation for enhanced recombineering and programmable antigen-responsive genome editing systems for therapeutic applications. It is my hope that this work will provide the foundation to better understand the eMAGE mechanism and guide the development of more advanced cellular control systems in the future.

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