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 isogenic cell lines that differ from each other by sequence variations introduced by genome editing, as we described for an iPSC-based model of Barth Syndrome3. The high efficiency of the procedure can also be used to simultaneously disrupt multiple genes or sequences that are present multiple times in the genome. For example, we used this strategy to simultaneously disrupt 62 copies of porcine endogenous retrovirus in a porcine cell line5. The inducibility of Cas9 in our system might also be exploited to permit temporally controlled gene inactivation in cells differentiated from iPSC, thereby potentially circumventing the need to establish stable mutant cell lines.
