Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs

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.

Analysis of Endothelial-to-Haematopoietic Transition at the Single Cell Level identifies Cell Cycle Regulation as a Driver of Differentiation

“…Plasmid carrying an inducible shRNA was generated as previously described (66). Briefly, shRNA for the CDK1 gene (shCDK1) was obtained from previous publication (67) and introduced by cloning of annealed oligonucleotides in the sOPTiKD plasmid between the BglII and SalI-HF sites.…”

“…The resulting sOPTiKD_shCDK vector was targeted to the AAVS1 locus in combination with the pZFN.AAVS1-KKR and pZFN.AAVS1-ELD vectors (66). Single cells hPSCs were nucleofected using the Lonza P3 Primary Cell 4D-Nucleofector X Kit and the cycle CA-137 on a Lonza 4D-Nucleofector System, in feeder-free conditions.…”

Cell cycle regulators control mesoderm specification in human pluripotent stem cells

“…Generation of CDK1 inducible knockdown line -Knockdown for CDK1 was performed using the single optimised inducible knockdown method as previously described (24,53). Multiple shRNAs for the CDK1 gene were obtained from the validated shRNA database at Sigma-Aldrich.…”

The End of Snoring? Application of CRISPR/Cas9 Genome Editing for Sleep Disorders

“…Thus, inducible loss of gene function experiments is necessary to uncover mechanisms underlying development, physiology and disease [69]. One of these gene editing techniques is known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).…”