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Updated: Jun 17, 2025
Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9
Published on: August 25, 2018
Protocol for establishing inducible CRISPRd system for blocking transcription factor-binding sites in human
Satoshi Matsui1, Joseph R Shiley1, Morgan Buckley1
1Division of Developmental Biology, Center for Stem Cell & Organoid Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, OH 45229, USA.
None:
Transcription factor (TF) gene knockout or knockdown experiments provide comprehensive downstream effects on gene regulation. However, distinguishing primary direct effects from secondary effects remains challenging. To assess the direct effect of TF binding events, we present a protocol for establishing a doxycycline (Dox)-inducible CRISPRd system in human pluripotent stem cells (hPSCs). We describe the steps for establishing CRISPRd host hPSCs, designing and preparing single-guide RNA (sgRNA) expression lentivirus vectors, generating CRISPRd hPSCs transduced with sgRNAs, and analyzing CRISPRd TF-block effects by chromatin immunoprecipitation (ChIP)-qPCR. For complete details on the use and execution of this protocol, please refer to Matsui et al.1.
