We then sought to exemplify the use of OPTiKD and sOPTiKD to rapidly and efficiently evaluate endogenous gene function in a variety of cell types and at different stages of hPSC differentiation related to embryonic development. First, we focused on the master developmental regulator T (brachyury), which plays an essential role in mesoderm formation and, in particular, during the development of posterior mesoderm, notochord and somites (Martin, 2015; Papaioannou, 2014). Indeed, mice carrying a heterozygous mutation in T exhibit a short tail phenotype, while homozygous mutations are embryonic lethal at around 9.5 dpc (Dobrovolskaia-Zavadskaia, 1927; Gluecksohn-Schoenheimer, 1944). T mutants also present severe cardiovascular and placental defects (David et al., 2011; Inman and Downs, 2006; King et al., 1998). Furthermore, T was recently shown to specifically regulate mesoderm but not endoderm differentiation in hPSCs (Faial et al., 2015).
