CRISPR/Cas9 Editing of DMD-iPSCs

The DMD-iPSCs were nucleofected with the Human Stem Cell Nucleofector^® Kit 2 (LONZA, Walkersville, MD, USA) set at program B016 according to the manufacturer’s instructions. Briefly, the DMD-iPSCs were dissociated into single cells using TrypLE™ Express (Thermo Fisher Scientific) and counted. For 1.5 × 10⁶ cells, 5 µg of CRISPR/Cas9 plasmid and donor plasmid were used, respectively, to transfect the DMD-iPSCs, then the transfected cells were cultured in mTesR1 medium with 10 µM of Y27632 (STEMCELL Technologies). Two days later, 50 µg/mL of G418 (Sigma-Aldrich) was used for screening the gene targeted cells. The cells survived from the G418 selection were counted, and 1000 cells were seeded on Matrigel-coated 6 cm dishes and cultured in mTesR1 medium with CloneR (STEMCELL Technologies) for 8 to 10 days. Then individual clones were picked, expanded and screened by PCR.
To analyze the potential off-target effect of CRISPR/Cas9, the predicted off-target sites with Cas-OFFinder [47 (link)], which is accessible at http://www.rgenome.net. (accessed on 1 June 2021), were detected via Sanger sequencing. We isolated gDNA of hiPSCs and Rn14-iPSCs for the PCR amplification and Sanger sequencing of the top15 potential off-target sites.