Aside from the examples reported in this manuscript, we envision several other potential applications of the sOPTiKD/KO technologies. With regard to cellular and developmental biology, we anticipate that sOPTiKO could efficiently accommodate variants of the Cas9 gene with catalytically inactive domains (Dominguez et al., 2015). For instance, Cas9 fusion proteins with epigenetic modifiers could allow functional validations of putative genomic regulatory regions. Similarly, sOPTiKD could be repurposed to drive other types of inducible non-coding RNAs, such as antagomir or miRNA sponges to probe microRNA function (Ebert and Sharp, 2010). Remarkably, the high targeting efficiency and scalability of sOPTiKD/KO could allow high-throughput screenings by targeting inducible shRNA or gRNA pools. Compared with viral-based approaches (Chen et al., 2012), the isogenic integration of inducible shRNAs/gRNAs would reduce heterogeneity in the targeted population, hence increasing the screening sensitivity and specificity. With regard to disease modeling applications, sOPTiKD/KO could allow the simultaneous targeting of several hiPSC lines to probe gene function in different genetic backgrounds. Such an approach could facilitate the identification of genetic disease modifiers and the discovery of novel potential drug targets
