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 (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development.
Highlights
Loss-of-function experiments in human pluripotent stem cells [hPSCs; comprising human embryonic stem cells or human induced pluripotent stem cells] provide a unique opportunity to study the mechanisms that regulate human development, physiology and disease (Avior et al, 2016; Pourquié et al, 2015; Zhu and Huangfu, 2013)
The AAVS1 and ROSA26 loci appeared suitable for this purpose as these genomic safe harbor (GSH) have been suggested to allow strong expression of various transgenes in hPSCs, including constitutively expressed short hairpin RNAs (shRNAs) (DeKelver et al, 2010; Hockemeyer et al, 2009; Irion et al, 2007)
HPSC targeting occurred with very high efficiency (59-100%; Table S1), while neither ROSA26 nor AAVS1 modifications resulted in chromosomal abnormalities
Summary
Loss-of-function experiments in human pluripotent stem cells [hPSCs; comprising human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs)] provide a unique opportunity to study the mechanisms that regulate human development, physiology and disease (Avior et al, 2016; Pourquié et al, 2015; Zhu and Huangfu, 2013). The expression of inducible short hairpin RNAs (shRNAs) has been the most popular method to trigger gene knockdown in human cells This has been achieved using a TETON system, which relies on a modified RNA polymerase (Pol) III promoter that is responsive to a tetracycline-sensitive repressor protein (tetR) to induce shRNA expression by simple tetracycline (TET) treatment (Lambeth and Smith, 2013). Application of this TET-ON system in hPSCs has proved challenging for two main reasons: (1) tight control of shRNA expression is difficult to achieve, thereby resulting in uncontrolled knockdown; (2) induction of shRNA rarely works in differentiated derivatives. Inducible shRNA expression in both hPSCs and a wide variety of their differentiated progenies has never been reported
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