Optimized inducible shRNA and CRISPR/Cas9 platforms for $\textit{in vitro}$ studies of human development using hPSCs
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Abstract
Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (
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1477-9129
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European Commission (278955)
Qatar Foundation for Education, Science and Community Development (unknown)
British Council in Israel (52BX14MKSR)
Wings for Life Spinal Cord Research Foundation (WFL-UK-005/11)
Medical Research Council (MR/L016761/1)
Medical Research Council (MC_PC_12009)
British Heart Foundation (None)