Genomic Tools to Decode and Engineer Gene Regulation

Abstract

Gene regulation is a complex process that defines cellular identity and function from a single set of DNA-encoded rules. At the cellular level, gene regulation determines how one genome can produce the diversity of cell types that form complex tissues and organs. At the sequence level, single-base changes in DNA can profoundly affect gene function and regulation, influencing cellular behavior and disease development. Understanding gene regulation at both scales—the macro and the micro—provides insights into how complex biological systems are built and maintained and how minute genetic variations can disrupt these systems, leading to disease. My research aims to develop methods for characterizing, perturbing, and engineering gene regulatory mechanisms that bridge both of these scales. In Chapter 1, I describe the development of a platform called HACE (Helicase-Assisted Continuous Editing) for performing targeted mutagenesis on endogenous mammalian genomes, allowing us to functionally characterize both coding and non-coding genomic elements. In Chapter 2, I present SPICE (Splicing Proportions In Cell Types), an integrated experimental and computational framework that harnesses alternative RNA splicing as a programmable modality for cell type--specific gene regulation. Using SPICE, I designed and engineered splicing elements that are specific to different cell types. In Chapter 3, I describe RADARS (Reprogrammable ADAR Sensors), another platform for cell type-- and cell state--specific gene regulation by sensing endogenous RNA transcripts using ADAR-dependent RNA editing. Together, these approaches establish a framework for decoding regulatory logic and engineering gene regulation with cell type specificity.