Human pluripotent stem cells (hPSCs) are emerging as an attractive model for studying human development and disease. However, functional studies of these cells are limited due to a lack of efficient methods for manipulating their gene expression. Here, Alessandro Bertero and co-workers devise platforms that allow for the inducible knockdown or knockout of specific genes in hPSCs and their derivatives (p. 4405). They first validate the ROSA26 and AAVS1 loci as genomic safe harbours that can be engineered in hPSCs to support stable transgene expression in a large panel of mature cells obtained from hPSCs. The authors then develop single-step optimized inducible knockdown (sOPTiKD) – an inducible shRNA-mediated approach for gene knockdown. This method allows for strong inducible expression of shRNAs, resulting in efficient gene knockdown even following hPSC differentiation. It also uses an optimized tetracycline-responsive repressor protein that eliminates leaky shRNA expression. Importantly, the authors show that this method can be used to knock down individual and multiple genes to study developmental mechanisms. They also develop a conditional knockout system based on CRISPR/Cas9 technology, named single-step optimized inducible knockout (sOPTiKO), and show that gene knockout using this system is possible in hPSCs and mature cell types. Given their robustness, high efficiency and scalability, these platforms promise to be valuable tools for the field.
