WB original data (Efficient exon skipping by base editor-mediated abrogation of exonic splicing enhancers)
Duchenne muscular dystrophy (DMD) is a severe genetic disease caused by the loss of the dystrophin protein. Exon skipping is a promising strategy to treat DMD by restoring truncated dystrophin. 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, we successfully identify novel ESEs in DMD exons 51 and 53. TAM-CBE induces near-complete skipping of the respective exons by targeting these ESEs in patients’ iPSCs-derived cardiomyocytes. Combined with strategies to disrupt splice sites, we identify suitable sgRNAs with TAM-CBE to efficiently skip most DMD hotspot exons without substantial double-stranded breaks. Our study thus expands the repertoire of potential targets for CBE-mediated exon skipping in treating DMD and other RNA mis-splicing diseases.
Steps to reproduce
WT, N040006(patient), E50-N040006(patient & ΔE50), TAM-corrected-rep1 to TAM-corrected-rep2 (patient & ΔE50&51) cardiomyocytes which differentiated from iPSCs for 30 days were lysed and prepared protein samples. anti-Dystrophin (Sigma, D8168) and anti-mouse-IgG-HRP, Rabbit polyclonal to Cardiac Troponin T (Abcam, Ab45932), Rabbit monoclonal to Vinculin (Abcam, Ab129002) and anti-Rabbit-IgG-HRP were used to detect the target protein respectively. GEL Imaging System (AI680RGB) were used to detect the protein band, and analyzed by ImageJ.