Finally, we explored whether sOPTiKD could enable simultaneous knockdown of multiple genes (Fig. 3E). We focused on the cyclin D family (CCND1, CCND2 and CCND3). These cell cycle regulators are functionally redundant, and thus their study in hESCs has previously required laborious multiple rounds of stable shRNA transfection in order to achieve double or triple knockdown (Pauklin and Vallier, 2013). We developed a method to easily combine multiple shRNAs into the same targeting vector using a one-step Gibson assembly, and generated sOPTiKD plasmids carrying one, two or three shRNAs against cyclin D genes or scrambled control shRNAs (Fig. 3E). These vectors were tested in hESCs without isolation of clonal sublines, and inducible knockdown proved highly efficient and comparable with single, double and triple shRNA constructs (Fig. 3F). Interestingly, prolonged knockdown of one or two cyclin Ds was compatible with hESC self-renewal (Fig. 3G), whereas triple knockdown resulted in definitive endoderm differentiation (Fig. 3G,H), as previously reported (Pauklin and Vallier, 2013; Pauklin et al., 2016). Collectively, these results demonstrate that sOPTiKD can be used to simultaneously decrease the expression of several genes with redundant functions.
