Efficient exon skipping by base editor-mediated abrogation of exonic splicing enhancers

Duchenne muscular dystrophy (DMD) is a fatal genetic disease caused by a completeloss of the dystrophin protein (coded by the DMD gene). Because internal truncateddystrophins are partially functional, exon skipping is a promising therapeutic approachto restoring truncated dystrophin and alleviating muscular dystrophies in patients withDMD. Recently, we have developed a cytidine base editor (CBE)--based technology togenetically modulate mRNA splicing and treat DMD by precisely inducing G>Aconversions at exon-intron junctions in genomic DNA. Here, we demonstrate that baseeditors (e.g., Targeted AID-mediated mutagenesis (TAM)) are able to efficiently induceexon skipping by disrupting functional redundant exonic splicing enhancers (ESEs). Bydeveloping an unbiased and high-throughput screening to interrogate exonicsequences at the nucleotide resolution, we successfully identified novel ESEs in DMDexons 51 and 53. TAM-CBE induced near-complete skipping of the respective exonsby targeting these ESEs. Combined with strategies to disrupt splice sites, we identifiedsuitable sgRNAs with TAM-CBE to efficiently skip most DMD hotspot exons withoutsubstantial double-stranded breaks in iPSC-derived cardiomyocytes. Our study thusexpands the repertoire of potential targets for CBE-mediated exon skipping in treatingDMD and other RNA mis-splicing diseases.