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

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.

SUBMITTER: Matsui S

PROVIDER: S-EPMC11357809 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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

Matsui Satoshi S Shiley Joseph R JR Buckley Morgan M Lim Hee-Woong HW Hu Yueh-Chiang YC Mayhew Christopher N CN Iwafuchi Makiko M

STAR protocols 20240811 3

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) ...[more]

PMID: 39133612

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