Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused 
by a mutation in the dystrophin gene. Genetic correction of patient-derived 
induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise 
for DMD gene therapy; however, the safety of such nuclease treatment must be 
determined. Using a unique k-mer database, we systematically identified a unique 
target region that reduces off-target sites. To restore the dystrophin protein, 
we performed three correction methods (exon skipping, frameshifting, and exon 
knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most 
effective approach. We further investigated the genomic integrity by 
karyotyping, copy number variation array, and exome sequencing to identify 
clones with a minimal mutation load. Finally, we differentiated the corrected 
iPSCs toward skeletal muscle cells and successfully detected the expression of 
full-length dystrophin protein. These results provide an important framework for 
developing iPSC-based gene therapy for genetic disorders using programmable 
nucleases.