Historically, the expression of inducible short hairpin RNAs (shRNAs) has been the most popular method to trigger gene knockdown in human cells. This has been achieved using a TET-ON system, which relies on a modified RNA polymerase (Pol) III promoter that is responsive to a tetracycline-sensitive repressor protein (tetR) to induce shRNA expression by simple tetracycline (TET) treatment (Lambeth and Smith, 2013). Nevertheless, application of this TET-ON system in hPSCs has proved challenging for two main reasons: (1) tight control of shRNA expression is difficult to achieve, thereby resulting in uncontrolled knockdown; (2) induction of shRNA rarely works in differentiated derivatives. Indeed, very high and homogenous expression of both the tetR and the inducible shRNA is required to obtain potent yet controlled knockdown. However, transgene silencing is a recurring problem in hPSCs (Ellis, 2005; Herbst et al., 2012; Yao et al., 2004), and randomly integrated promoters are often subject to positional effects that can strongly limit their activity (Zafarana et al., 2009). Differentiation further increases the chances of silencing, as transgenes can be located in regions where heterochromatin forms following
