Date of Graduation

5-2024

Document Type

Thesis

Degree Name

Bachelor of Science in Biomedical Engineering

Degree Level

Undergraduate

Department

Biomedical Engineering

Advisor/Mentor

Nelson, Christopher

Abstract

Gene replacement is a promising method of therapy for genetic diseases. However, safety and efficacy are areas that need more research. This experiment aims to use RNA sequencing and bioinformatic techniques to provide answers to these questions and provide direction to future studies to develop a gene replacement therapeutic. C2C12 mouse myoblast cells were transfected with a vector containing a CRISPR-Cas9 system and the Human Factor IX (hF9) gene in order to hijack target genes and integrate the hF9 gene. The two target genes, myoglobin (Mb) and creatine kinase (Ckm), were chosen for their high rate of expression and low impact on survival when removed in vivo. RNA sequencing was performed to analyze the transcriptome of the mouse cells using differential expression analysis and splicing isoform analysis. Differential expression revealed Mb was downregulated and Ckm was not significantly affected, while some other genes had significant changes in their expression rate. Splicing isoform analysis revealed that the hF9 gene was integrated into the mouse genome but had variable expression along different points along the gene. Overall, this experiment and analysis techniques show promise in using the Mb and Ckm sites for gene replacement. However, experimentation is needed to find the cause and in vivo effects of expression changes in off-target genes.

Keywords

CRISPR; Gene Replacement; Gene Therapy; RNA Sequencing; Bioinformatics; Genome Engineering

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Available for download on Saturday, April 25, 2026

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