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 is achieved either by transient transfection, which can be poor in efficiency, or by random integration of the gRNA, which can result in mosaic expression. Finally, whereas CRISPR interference can only efficiently control gene promoter activity (Mandegar et al., 2016), sOPTiKO allows the deletion of a broader range of genomic targets, including regions outside of promoters that might not have a direct role in transcriptional regulation. Overall, sOPTiKD/KO are the first inducible shRNA and CRISPR/Cas9 technologies that enable streamlined functional studies of multiple genetic variants in hPSCs and in a diversity of differentiated cell types (Fig. 8G).
