Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of inducible gene expression.

Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to; 1) exons of an endogenous gene of interest (GOI) and 2) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression. Transcriptome and ChIP-seq analyses of inducible CDX2 integrated expression and wt CDX2 expression in LS174T colorectal cell lines by deep sequencing. In total six transcriptome data set (LS174T_wt; LS174T_CDX+/-; LS174T_CDX2-/-; LS174T_CDX2-/-1X induced CDX2 (4ng Dox); LS174T_CDX2-/-2X induced CDX2 (0ng Dox); LS174T_CDX2-/-2X induced CDX2; 4ng Dox); LS174T_CDX2-/-2X induced CDX2 (40ngDox)) and one ChIP-seq data set (ChIP-seq CDX2 ab)

基于四环素的诱导系统为可精准调控基因表达的功能研究提供了强有力的技术手段。然而，该诱导系统存在调控严谨性不足的缺陷，易因四环素剂量使用不当引发泄漏表达及不良反应，这已成为其应用局限。本研究报道，将基因组编辑工具与新一代Tet-On诱导系统联用，可有效解决上述问题。本策略基于将诱导转录元件（命名为PrIITE）精准整合至两个靶点：1）目标内源基因（gene of interest, GOI）的外显子区域；2）安全港位点。我们利用携带GFP报告基因或CDX2转录因子的PrIITE细胞，证实该系统可实现基因表达的严格诱导，且完全消除泄漏表达。通过本方法构建的CDX2 PrIITE细胞，揭示了全新的CDX2下游效应基因。本研究结果为解析必需基因的剂量依赖性效应功能提供了可行策略，此类研究需确保内源基因表达完全缺失。本研究通过深度测序，对LS174T结直肠细胞系中整合型诱导性CDX2表达与野生型（wild type, wt）CDX2表达开展转录组与染色质免疫沉淀测序（ChIP-seq）分析。共计6组转录组数据集：LS174T_wt、LS174T_CDX+/-、LS174T_CDX2-/-、LS174T_CDX2-/-1X诱导型CDX2（4ng 多西环素（Doxycycline, Dox））、LS174T_CDX2-/-2X诱导型CDX2（0ng Dox）、LS174T_CDX2-/-2X诱导型CDX2（4ng Dox）以及LS174T_CDX2-/-2X诱导型CDX2（40ng Dox）；另有1组ChIP-seq数据集（ChIP-seq CDX2抗体）。