Successful In Vitro Modification of the Dmd Gene Using Prime Editing

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.