Graduation Year

2025

Document Type

Campus Only Senior Thesis

Degree Name

Bachelor of Arts

Department

Biology

Reader 1

Kyle Jay

Reader 2

Marcus Noyes

Abstract

Retinitis pigmentosa (RP) encompasses a heterogeneous array of inherited diseases that lead to progressive deterioration of retinal photoreceptors, resulting in gradual loss of vision. Mutations in the Rhodopsin (RHO) gene frequently lead to autosomal dominant RP (adRP), for which effective therapeutic treatments have not yet been developed. Specifically, the Pro23His (P23H) mutation in RHO has been documented as the most common RHO mutation that leads to a gain- of-function effect, accounting for about 12% of adRP cases. Here, we aim to engineer allele- specific Cys2His2 zinc fingers (ZF) to repress the RHO-P23H gain-of-function mutation without impacting the healthy allele. We screened novel 2-finger P23H-specific constructs predicted by the machine learning model, ZFDesign, using a bacterial one-hybrid (B1H) system. After selecting the 2-finger constructs with the highest allele specificity, 5-finger constructs were further screened using fluorescence-activated cell sorting to determine ZF discrimination between the mutant P23H and the wildtype allele. Illumina sequencing of recovered ZF candidates identified mutants with strong RHO-P23H specificity, achieving on-target sequence enrichment exceeding 0.90 while maintaining critical off-target enrichment below 0.1. The development of allele-specific ZFs to regulate the RHO-P23H mutation in rhodopsin has great implications for novel gene therapeutics to address adRP in humans.

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