Fig. 1. Summary of the key steps of CRISPR/Cas9-mediated genome editing in iPSCs.

Overall, it only takes 2 weeks to generate individual clones after CRISPR/Cas9-mediated gene knockout. After electroporation, cells were treated with puromycin for a single day to remove nontransfected cells, and then the surviving cells with GFP fluorescence were recovered in normal medium without puromycin. Because this method relies on the transient endonuclease activity of the gRNA/Cas9 complex to induce double-strand breakage at the genomic target site, this method is focused on the short-term expression of gRNA/Cas9 in puromycin medium followed by dilution of the intracellular plasmids in normal medium along with further propagation.