Seamless genome editing in human pluripotent stem cells using custom endonuclease-based gene targeting and the piggyBac transposon.

I report here a detailed protocol for seamless genome editing using the piggyBac transposon in human pluripotent stem cells (hPSCs). Recent advances in custom endonucleases have enabled us to routinely perform genome editing in hPSCs. Conventional approaches use the Cre/loxP system that leaves behind residual sequences in the targeted genome. I used the piggyBac transposon to seamlessly remove a drug selection cassette and demonstrated safe genetic correction of a mutation causing α-1 antitrypsin deficiency in patient-derived hPSCs. An alternative approach to using the piggyBac transposon to correct mutations involves using single-stranded oligonucleotides, which is a faster process to complete. However, this experimental procedure is rather complicated and it may be hard to achieve homozygous modifications. In contrast, using the piggyBac transposon with drug selection-based enrichment of genetic modifications, as described here, is simple and can yield multiple correctly targeted clones, including homozygotes. Although two rounds of genetic manipulation are required to achieve homozygote modifications, the entire process takes ∼3 months to complete.