Although Cas9 efficiently directs target site cleavage, the efficiency of targeted genome modification was initially reported to be approximately 1–2% in human iPSCs7,9. At this efficiency, recovery of properly targeted clones without positive selection is labor intensive and inconsistent. We and others previously showed that low transfection efficiency of the relatively large Cas9 expression construct limits yield of targeted clones in pluripotent stem cells10. Procedures that select for Cas9 transfected cells, such as cell sorting for a fluorescent protein expressed from a co-transfected plasmid, increase the recovery of modified clones11. However, cell sorting is stressful for stem cells, exposes them to contamination risks, and can be cumbersome when performing modifications on multiple cell lines in parallel. Gonzalez et al. showed that knockin of inducible Cas9 into a safe harbour locus enhances genome editing efficiency12. However, this strategy consumed the AAVS safe harbour locus and the Cas9 transgene was not exciseable. We have refined this strategy and present here an optimized protocol to permit footprint-free, highly efficient and consistent genome modification in human iPSCs. This procedure can be used to develop
