Ayesha Siddika

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish dystrophin expression. Prime editing enables precise genome modification without generating double-strand DNA breaks or requiring donor DNA templates. We established an in vitro prime editing workflow to generate and subsequently correct the mdx5cv mutation in mouse C2C12 myoblasts. Following optimization of engineered prime editing guide RNAs (epegRNAs) and PAM-flexible prime editors, wild-type cells were edited, clonally isolated, and genotyped. Mutation correction was then evaluated using optimized epegRNA designs. Two rounds of prime editing introduced the mdx5cv mutation into approximately 20% of alleles in C2C12 cells creating the mdx5cv C2C12 cell line. Clonal isolation yielded five homozygous mutant clones among 59 expanded colonies. Optimization studies identified an epegRNA containing a 16 nucleotide reverse transcription template and a 10 nucleotide primer binding site (RTT16/PBS10) as the most efficient design. Correction of the pathogenic allele reached approximately 26%, whereas longer PBS lengths reduced editing efficiency. In silico off-target analysis using Cas-OFFinder identified no candidate genomic loci with fewer than three mismatches for the spacer sequences used in either mutation generation or correction, suggesting a favorable predicted specificity profile. Corrected mdx5cv C2C12 myoblasts retained their capacity to differentiate into multinucleated myotubes. Representative Western blot analysis detected dystrophin protein expression in differentiated corrected mdx5cv myotubes, consistent with successful correction of the pathogenic mutation. These findings establish a robust prime editing platform for both the generation and correction of the mdx5cv mutation and provide proof of concept that precise correction of the pathogenic mutation is associated with restoration of dystrophin expression following myogenic differentiation.

Duchenne muscular dystrophy (DMD) remains a major challenge in genetic medicine due to the difficulty of achieving durable, body-wide restoration of dystrophin in post-mitotic muscle tissues. Although current therapies—including exon skipping and micro-dystrophin gene replacement—have demonstrated clinical feasibility, their benefits are limited by incomplete efficacy, mutation specificity, and the need for repeated or high-dose interventions. These limitations highlight the need for strategies capable of directly and permanently correcting the underlying genetic defect. Recent advances in genome editing have positioned CRISPR-based technologies as promising candidates for this objective. Rather than functioning as a single approach, gene-editing platforms encompass a spectrum of strategies—including exon deletion, exon reframing, base editing, and prime editing—each with distinct advantages depending on the mutational context. In particular, the emergence of precision editing tools has enabled controlled nucleotide-level modifications, expanding the range of correctable mutations while reducing reliance on double-strand DNA breaks. In this review, we adopt a comparative and translational perspective to evaluate gene-editing strategies for DMD. We examine how different approaches align with specific mutation types, summarize key findings from preclinical studies, and analyze the major barriers to clinical implementation, including delivery efficiency, immune responses, editing durability, and genomic safety. We further discuss emerging innovations in editing technologies and delivery systems that aim to address these limitations. Collectively, this work reframes gene editing as a decision-oriented and application-driven therapeutic framework. Continued integration of advances in genome engineering, delivery platforms, and muscle biology will be essential to translate these technologies into safe, effective, and durable treatments capable of altering the clinical trajectory of DMD.

Duchenne muscular dystrophy (DMD) is a fatal X-linked neuromuscular disorder caused by mutations in the dystrophin gene. Prime editing is a versatile genome editing technology capable of introducing precise nucleotide changes without generating double-strand DNA breaks, making it a promising approach for correcting pathogenic point mutations. In this study, we applied prime editing to modify mdx-4cv and mdx-5cv mutation-equivalent sites in mouse C2C12 myoblasts in vitro. Initial editing efficiencies were unexpectedly low and were associated with the presence of a 5′-TTCT-3′ motif within engineered prime editing guide RNAs (epegRNAs). epegRNA designs containing this motif exhibited reduced prime editing efficiency, whereas silent substitution eliminating the motif significantly improved editing outcomes, indicating that specific sequence features within epegRNAs can influence editing performance. Rational redesign of epegRNAs to remove this motif substantially enhanced editing efficiency, achieving up to 20% modification at the 4cv target site using an NGG PAM and 21% editing at the 5cv locus using an NGAG PAM. These findings highlight an important sequence-dependent constraint in epegRNA design and provide practical guidance for optimizing prime editing strategies targeting Dmd mutations in vitro.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, a genome-editing technology pioneered in 2012, enables the precise correction of deleterious mutations or disruption of disease-causing genes through targeted double-strand breaks (DSBs), offering potential for treating genetic diseases. However, CRISPR/Cas9 can cause off-target cleavage at non-specific DNA sites, leading to unintended insertions or deletions (indels), which limit its safety and applicability despite ongoing improvements in specificity. Recently, prime editing (PE), an advanced CRISPR-derived technology, has been employed with a Cas9 nickase (Cas9n) fused with a reverse transcriptase and a prime editing guide RNA (pegRNA) to enable precise insertions, deletions, and transversions without inducing DSBs, thus reducing risks of indels and chromosomal aberrations. Furthermore, ongoing optimizations, such as improved pegRNA design and enhanced editing efficiency, have expanded the applications of PE in medical therapeutics, agriculture, and fundamental research. This review summarizes recent advancements in the PE system, including optimized pegRNA designs and enzyme engineering for enhanced efficiency and specificity, alongside novel delivery methods. It also evaluates cutting-edge delivery strategies, such as adeno-associated virus (AAV) vectors, lipid nanoparticles (LNPs) and novel extracellular vesicle (EV)-based systems, and explores PE applications in vitro and in vivo, including disease modeling and therapeutic gene correction.

Duchenne muscular dystrophy (DMD) is a fatal X-linked neuromuscular disorder caused by mutations in the dystrophin gene. Prime editing is a versatile genome-editing technology capable of precise base correction without inducing double-strand DNA breaks, making it well suited for correction of point mutations in the DMD gene. Here, we applied prime editing to correct the mdx-4cv and mdx-5cv mutations in mouse myoblasts in vitro. Initial editing efficiencies were unexpectedly low and were traced to the presence of a 5′-TTCT-3′ motif within engineered prime editing guide RNAs (epegRNAs), consistent with premature RNA polymerase III–mediated transcription termination from the U6 promoter. Rational redesign of epegRNAs to eliminate this motif markedly improved editing efficiency, achieving up to 20% correction of the 4cv mutation and 21% editing at the 5cv locus using NGAG PAM. These results identify a critical epegRNA design constraint and establish an efficient prime editing framework for precise correction of DMD point mutations.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), an exceptionally potent genome-editing technique developed in 2012, is the ideal tool of the future for treating diseases by permanently correcting deleterious base mutations or disrupting disease-causing genes with great precision and efficiency. However, it is prone to cleaving double-stranded DNA in off-target genes and generating random mutations in the process. These drawbacks restrict its application in fundamental research and agriculture, and raises safety concerns in the field of medicine. Fortunately, the new gene editing technology derived from CRISPR/Cas9, known as prime editing, has the potential to provide targeted sequence insertion, deletion, and transversion, all while avoiding the formation of double-strand breaks, thus minimizing adverse effects. Meanwhile, the rapid development of this technology makes its application wider and broader. This review summarizes the current developments and optimizations of the prime editing (PE) system with improved editing efficiency and precision. Along with discussing the most recent delivery techniques and outlining the PE applications that are being used both in vitro and in vivo.