Date of Graduation
Master of Science in Biomedical Engineering (MSBME)
Christopher E. Nelson
Jeffery C. Wolchok
Second Committee Member
CRISPR-Cas9, DNA Sequencing, Duchenne Muscular Dystrophy, Gene Editing
This work establishes a method for assessing on-target precision due to CRISPR-Cas9 gene editing, especially within the context of exon skipping therapy for Duchenne Muscular Dystrophy. The proposed method utilizes an Oxford nanopore long-read sequencing approach to sequence amplified regions of DNA that have been edited using CRISPR-Cas9. NIH3T3 and C2C12 cell lines were treated with a dual-guide CRISPR-Cas9 system, that targets and deletes exon 23 from the DMD gene in mouse samples. Deletion PCR revealed deletion of exon 23 in both DNA and cDNA samples. Additionally, sequencing using Oxford Nanopore revealed targeted exon 23 deletion as the most prevalent event. There were additional editing events, including large deletions, insertions, pseudoexons, and a duplication. The next step is to sequence in vivo mouse samples that were treated with the same dual-guide system targeting exon 23. This will reveal additional unintended editing events such as vector integration and will provide further insight into the efficacy of CRISPR-based therapies. Future work will focus on optimizing an amplification-free method for target enrichment and long-read sequencing.
Burcham, L. A. (2022). Development of a Long-Read Sequencing Protocol to Assess the Precision and Efficacy of Gene Editing for Duchenne Muscular Dystrophy. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/4446