Comprehensive long- and short- read sequencing approaches to screen the mutational landscape after therapeutic CRISPR-editing for Duchenne Muscular Dystrophy
Date of Graduation
Bachelor of Science in Biomedical Engineering
Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disease caused by mutations in the Dmd gene resulting in non-functional dystrophin. Adeno-associated virus (AAV)-mediated delivery of classical CRISPR systems resulting in double-stranded breaks (DSBs) has yielded promise as a curative DMD therapeutic. However, the consequences of AAV-CRISPR require more thorough characterization. PCR-enriched short-read amplicon sequencing at cut sites is the most common method of sequencing CRISPR editing, but PCR bias results in unreliable quantification of on-target edits. We analyze orthogonal long- and short-read sequencing approaches to screen the outcomes of CRISPR technologies at the DNA and RNA-level for DMD. We performed intramuscular injections of AAV-CRISPR or liposomes containing SpCas9 mRNA excising exon 23 using dual gRNAs at intron 22 and 23, in mdx and wild-type mice. Editing was confirmed using short-read indel sequencing. Nanopore sequencing of a 10kb PCR-enriched amplicon at the target site, in comparison, produces a broader catalog of editing events but similarly cannot avoid PCR-bias. We developed unbiased approaches for short- and long-read sequencing libraries with UMI’s and nanopore Cas9-targeted sequencing (nCATS) samples to compare these methods. We also examined the transcriptional impact of AAV-CRISPR on Dmd in mdx and wild-type mice. 5’ RACE nanopore libraries revealed AAV integration at the RNA-level in up to 0.3% of reads, creating an AAV-Dmd chimeric RNA with the transcriptional start site primarily originating downstream of the internal CMV promoter of the AAV genome. To further assess AAV-integration at the RNA-level, we performed 3’ RACE using the integrated AAV genome as the gene-specific primer to detect off-target locations of AAV integration and chimeric transcripts at the 3’ end. These strategies have been additionally replicated in mouse myoblasts using both AAV and electroporation edited samples to determine editing outcome differences in vitro and in vivo. We also used next-generation enrichment approaches to investigate prime editing-mediated exon skipping or targeted integration by PASTE for DMD in vitro. Our results indicate that double-stranded break-free methods are more precise but have a low efficiency at target sites. These results are significant for the translation of AAV-CRISPR for DMD and facilitates the development of unbiased sequencing approaches.
Duchenne Muscular Dystrophy, CRISPR, Next-gen sequencing, Adeno-associated viruses, Prime editing, PASTE
Jia, M., Padmaswari, M. H., Burcham, L., Agrawal, S., Ivy, A., Bryan, A., & Nelson, C. E. (2023). Comprehensive long- and short- read sequencing approaches to screen the mutational landscape after therapeutic CRISPR-editing for Duchenne Muscular Dystrophy. Biomedical Engineering Undergraduate Honors Theses Retrieved from https://scholarworks.uark.edu/bmeguht/137
Available for download on Tuesday, April 28, 2026
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