Protocol for establishing inducible CRISPRd system for blocking transcription factor-binding sites in human pluripotent stem cells

Abstract

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Cell culture
• Cell isolation
• Flow Cytometry
• Molecular Biology
• Chromatin immunoprecipitation