Having established an optimized inducible knockdown platform, we turned our attention to developing a complementary inducible knockout approach. Current inducible CRISPR/Cas9 methods rely on conditional overexpression of Cas9 in the presence of a constitutively expressed gRNA (González et al., 2014; Mandegar et al., 2016). In this case, control of Cas9 overexpression is achieved by a TET-ON method in which, following doxycycline treatment, a tetracycline-controlled reverse transactivator (rtTA) activates a Pol II-dependent tetracycline-responsive element (TRE) promoter (a fusion between multiple TET operons and a minimal CMV promoter). Although this TET-ON platform has been successfully applied to certain human cell types (Qin et al., 2010), we observed that this inducible system is silenced during hPSC differentiation into multiple lineages (including cardiomyocytes, hepatocytes and smooth muscle cells), even after targeting into the AAVS1 GSH (Fig. S7). These observations reinforce previous reports (Haenebalcke et al., 2013; Mandegar et al., 2016; Ordovas et al., 2015) and demonstrate that recently described systems for inducible CRISPR/Cas9 (González et al., 2014; Mandegar et al., 2016) are unlikely to work in a diversity of hPSC-derived cell types. For this
