Protocol for establishing inducible CRISPRd system for blocking transcription factor-binding sites in human pluripotent stem cells
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.<sup>1</sup>.
Authors
- Joseph R Shiley (ORCID: https://orcid.org/0000-0001-8453-1738)
- Yueh‐Chiang Hu (ORCID: https://orcid.org/0000-0002-7992-0525)
- Satoshi� Matsui (ORCID: https://orcid.org/0000-0002-6315-4109)
- Hee‐Woong Lim (ORCID: https://orcid.org/0000-0001-8599-1808)
- Morgan Buckley (ORCID: https://orcid.org/0009-0008-2015-7730)
- Makiko Iwafuchi (ORCID: https://orcid.org/0000-0003-1291-6246)
- Christopher N. Mayhew (ORCID: https://orcid.org/0000-0002-7408-1726)
Institutions
- Cincinnati Children's Hospital Medical Center (US)
- University of Cincinnati (US)
- University of Cincinnati Medical Center (US)
Publication Details
- Journal
- STAR Protocols
- Published
- 2024-08-11
- DOI
- https://doi.org/10.1016/j.xpro.2024.103233
- Citations
- 1
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 0.23
Funders
- Cincinnati Children's Hospital Medical Center
- Uehara Memorial Foundation
- Cincinnati Children’s Research Foundation
- Foundation for Promotion of Material Science and Technology of Japan
- National Institutes of Health
- Japan Society for the Promotion of Science