Having demonstrated that sOPTiKO allows efficient control of CRISPR/Cas9 activity in undifferentiated hPSCs, we thoroughly tested its performance following differentiation. We differentiated homozygous EGFPd2 inducible knockout cells carrying a single copy of inducible EGFP gRNA into the three primary germ layers and into five cell types of clinical interest (neurons, cardiomyocytes, smooth muscle cells, hepatocytes and endocrine pancreatic cells). Immunostaining for lineage-specific markers demonstrated that treatment with tetracycline resulted in strong loss of EGFPd2 expression (Fig. 8A-F, Fig. S9A,B) in all these cell types. Moreover, flow cytometry quantification confirmed that inducible knockout in differentiated cells was tightly controlled and efficient (Fig. S9C-H). For example, 85% of neuronal cells and 75% of smooth muscle cells completely lost EGFPd2 expression following tetracycline treatment (Fig. S9C,F). Considered together, these results validate that sOPTiKO allows efficient control of CRISPR/Cas9 activity not only in hPSCs, but also into a large panel of mature cell types (Fig. 8G).
