Date of Graduation

5-2025

Document Type

Thesis

Degree Name

Bachelor of Science in Biomedical Engineering

Degree Level

Undergraduate

Department

Biomedical Engineering

Advisor/Mentor

Nelson, Christopher

Abstract

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder that primarily affects boys. DMD causes a significant reduction in life expectancy due to progressive muscle deterioration through a mutation in the dystrophin protein. Gene editing techniques have been established as a way to combat the effects of DMD and have gradually shown progress in increasing the life expectancy for DMD individuals. A new gene editing technology, CRISPR-DREAM has the potential to revolutionize CRISPR technologies through its use of human mechanosensitive transcription factors as transactivation domains. This has shown promise in overcoming size limitations and toxicity associated with viral transactivation domains in current CRISPR platforms. This research aims to optimize the activation of CRISPR-DREAM through a lipofectamine-mediated time-course transfection. C2C12 mouse skeletal muscle cells were transfected with dCas9, DREAM, and cis-regulatory guide RNA plasmids and subsequently incubated for 24, 48, and 72 hours prior to RNA extraction and RT-qPCR analysis. Using RT-qPCR with primers specific to the CRISPR-DREAM plasmid, optimal transcriptional activation can be quantified. Once activation is sufficient for therapeutic relevance, CRISPR-DREAM can be used to increase the RNA expression of genes that are physiologically similar to dystrophin proteins. The protein products of these genes could be used to combat the detrimental effects of mutated dystrophin proteins.

Keywords

CRISPR-DREAM; Muscular Dystrophy; Transcriptional Upregulation; Dystrophin; Utrophin; Lipofectamine Transfection

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Available for download on Sunday, April 23, 2028

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