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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.
Abstract:
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.
Insights
This study introduces a new method using doxycycline-inducible CRISPR interference (CRISPRi) in human pluripotent stem cells (hPSCs) to precisely block transcription factor (TF) binding. This allows researchers to distinguish direct TF effects from complex secondary gene regulation changes.
Area of Science:
- Molecular Biology
- Stem Cell Biology
- Gene Regulation
Background:
- Transcription factor (TF) gene knockout/knockdown reveals downstream effects on gene regulation.
- Distinguishing primary direct TF effects from secondary effects is challenging.
- CRISPR interference (CRISPRi) offers a way to specifically block TF binding.
Purpose of the Study:
- To develop a protocol for assessing direct TF binding events.
- To establish a doxycycline (Dox)-inducible CRISPRi system in human pluripotent stem cells (hPSCs).
- To enable precise analysis of TF direct effects on gene regulation.
Main Methods:
- Established a doxycycline (Dox)-inducible CRISPRi system in hPSCs.
- Designed and prepared single-guide RNA (sgRNA) lentivirus vectors.
- Generated CRISPRi hPSCs transduced with sgRNAs.
- Analyzed CRISPRi TF-block effects using chromatin immunoprecipitation (ChIP)-qPCR.
Main Results:
- Successfully established a functional Dox-inducible CRISPRi system in hPSCs.
- Demonstrated the ability to specifically block TF binding events.
- Enabled the analysis of direct TF-mediated gene regulation.
Conclusions:
- The developed CRISPRi system provides a robust method for studying direct TF functions.
- This protocol facilitates the dissection of gene regulatory networks by isolating primary TF effects.
- The system is valuable for research in stem cell biology and developmental processes.
