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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.
摘要：
转录因子(TF)基因敲除或敲除实验提供了对基因调控的综合下游效应。然而,区分主要直接效应和次要效应仍然具有挑战性。为了评估TF结合事件的直接影响，我们提出了在人多能干细胞（hPSC）中建立多西环素（Dox）诱导型CRISPRd系统的方案。我们描述了建立CRISPRd宿主hPSC的步骤，设计和制备单向导RNA（sgRNA）表达慢病毒载体，产生用sgRNA转导的CRISPRdhPSC，并通过染色质免疫沉淀（ChIP）-qPCR分析CRISPRdTF阻断效应。有关此协议的使用和执行的完整详细信息，请参考松井等。1.
