Abstract
A central goal of genetics is to link genetic information to cellular phenotype, which often requires measuring it in individual living cells. Established approaches such as sequencing and hybridization-based imaging require lysing or fixing the cell, and so cannot track a dynamic process in a single living cell. This dissertation develops BroadCAS (Broad-range-genetic-information Colocalization-induced Amplifier of Signal), a programmable, modular platform for detecting genetic information in living cells: two target locators bind a feature side by side, so the presence of the feature becomes the colocalization of the two locators, which switches on a reporter that recruits the cell's transcription-translation machinery to amplify the signal into a single-cell readout. The platform comprises three independently optimizable modules, the target locator, the adaptor, and the reporter, and the work asks whether this design functions for RNA, whether its modules can be independently optimized and freely combined, whether a programmable Cas protein can serve as an RNA locator, and whether it generalizes beyond RNA. Chapter 2 establishes the Dual-Aptamer (DA) reporter, the non-programmable, reporter form of BroadCAS, as a non-disruptive RNA-monitoring system in which a self-contained MS2-PP7 aptamer structure recruits a two-hybrid detection module. Systematic engineering of multi-epitope arrays, split-inteins, a minimal-promoter substitution, and a Cre-loxP cascaded amplification circuit produced two application-tailored configurations, a High-Specificity Variant and a High-Sensitivity Variant; the optimizations added up only when they acted through independent mechanisms. The reporter quantified the long non-coding RNA LINC00355 in a breast-cancer model while largely preserving its native regulatory function. Chapter 3 extends the platform toward sensing native, unmodified transcripts by adapting catalytically inactive Cas13 (dCas13) as a reprogrammable RNA locator; this ongoing work screens guide-RNA pairs and Cas13 variants, compares strategies for linking effectors to dCas13, tests aptamer configurations, and introduces mutations that disable crRNA processing so that dCas13 stays compatible with the adaptor designs. Chapter 4 frames how the same colocalization principle could read DNA sequence variants, transcription-factor binding, and topological chromatin interactions. Together, the work advances a programmable, modular system that converts genetic information, beginning with RNA, into an amplified living-cell fluorescent readout, demonstrated as a reporter and extended toward a sensor.
Committee Chair
Gabriel Haller
Committee Members
Jessica Silva-Fisher; John Edwards; Robi Mitra; William Buchser
Degree
Doctor of Philosophy (PhD)
Author's Department
Biology & Biomedical Sciences (Molecular Genetics & Genomics)
Document Type
Dissertation
Date of Award
8-17-2026
Language
English (en)
DOI
https://doi.org/10.7936/mcnd-8396
Recommended Citation
Wang, Geng, "Engineering Modular RNA Detection Systems in Living Cells: From Dual-Aptamer Reporters Toward a Programmable Sensor for Diverse Genetic Information" (2026). Arts & Sciences Graduate Student Theses and Dissertations. 3872.
The definitive version is available at https://doi.org/10.7936/mcnd-8396