This report describes sOPTiKD and sOPTiKO – two novel platforms for inducible knockdown or knockout of gene expression that address the limitations of previous methods. Compared with alternative approaches that rely on viral transduction or random integration of inducible shRNAs (Lambeth and Smith, 2013; Zafarana et al., 2009), sOPTiKD is simpler to use (plasmid based), quicker (2 weeks or less to generate stable lines following a single step of gene targeting by lipofection), more efficient (>95% of the resulting cells show inducible knockdown), more scalable (isolation of clonal sublines can be entirely bypassed) and significantly more robust (due to the use of GSHs and the lack of leakiness). Furthermore, sOPTiKO shares these same advantages, thus outperforming recent inducible CRISPR/Cas9 methods that rely on the conditional expression of Cas9 (González et al., 2014) or of a fusion protein between a catalytically inactive Cas9 and the transcriptional repressor KRAB (Mandegar et al., 2016). Indeed, these systems rely on the TRE promoter, which is heavily silenced upon hPSC differentiation into multiple lineages. Furthermore, these are lengthy two-step methods, and expression of the gRNA
