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

Duchenne muscular dystrophy (DMD) is a fatal genetic disease caused by a complete loss of the dystrophin protein (coded by the DMD gene). Because internal truncated dystrophins are partially functional, exon skipping is a promising therapeutic approach to restoring truncated dystrophin and alleviating muscular dystrophies in patients with DMD. Recently, we have developed a cytidine base editor (CBE)--based technology to genetically modulate mRNA splicing and treat DMD by precisely inducing G>A conversions at exon-intron junctions in genomic DNA. Here, we demonstrate that base editors (e.g., Targeted AID-mediated mutagenesis (TAM)) are able to efficiently induce exon skipping by disrupting functional redundant exonic splicing enhancers (ESEs). By developing an unbiased and high-throughput screening to interrogate exonic sequences at the nucleotide resolution, we successfully identified novel ESEs in DMD exons 51 and 53. TAM-CBE induced near-complete skipping of the respective exons by targeting these ESEs. Combined with strategies to disrupt splice sites, we identified suitable sgRNAs with TAM-CBE to efficiently skip most DMD hotspot exons without substantial double-stranded breaks in iPSC-derived cardiomyocytes. Our study thus expands the repertoire of potential targets for CBE-mediated exon skipping in treating DMD and other RNA mis-splicing diseases.