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

Autor:	Matsui S; Division 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. Electronic address: matsui.satoshi.3p8@osaka-u.ac.jp., Shiley JR; Division 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., Buckley M; Division 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., Lim HW; Department of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, OH 45229, USA., Hu YC; Division 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., Mayhew CN; Division 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. Electronic address: christopher.mayhew@cchmc.org., Iwafuchi M; Division 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. Electronic address: makiko.iwafuchi@cchmc.org.
Jazyk:	angličtina
Zdroj:	STAR protocols [STAR Protoc] 2024 Sep 20; Vol. 5 (3), pp. 103233. Date of Electronic Publication: 2024 Aug 11.
DOI:	10.1016/j.xpro.2024.103233
Abstrakt:	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 . Competing Interests: Declaration of interests The authors declare no competing interests. (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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