Tools capable of modulating gene expression in living organisms are very useful for interrogating the gene regulatory network and controlling biological processes. The catalytically inactive CRISPR/Cas9 (dCas9), when fused with repressive or activating effectors, functions as a versatile platform to reprogram gene transcription at targeted genomic loci. However, without temporal control, the application of these reprogramming tools will likely cause off-target effects and lack strict reversibility. To overcome this limitation, we report herein the development of a chemical or light-inducible transcriptional reprogramming device that combines photoswitchable genetically encoded calcium actuators with dCas9 to control gene expression. By fusing an engineered Ca²⁺-responsive NFAT fragment with dCas9 and transcriptional coactivators, we harness the power of light to achieve photoinducible transcriptional reprogramming in mammalian cells. This synthetic system (designated CaRROT) can also be used to document calcium-dependent activity in mammals after exposure to ligands or chemicals that would elicit calcium response inside cells.

中文翻译：

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重新连接钙信号以实现精确的转录重编程

能够调节生物体中基因表达的工具对于询问基因调控网络和控制生物过程非常有用。无催化活性的 CRISPR/Cas9 (dCas9) 与抑制性或激活性效应器融合时，可作为多功能平台在目标基因组位点重编程基因转录。然而，如果没有时间控制，这些重编程工具的应用可能会导致脱靶效应并且缺乏严格的可逆性。为了克服这一限制，我们在此报告了一种化学或光诱导转录重编程装置的开发，该装置将光可切换的基因编码钙致动器与 dCas9 相结合以控制基因表达。通过融合工程化的 Ca ²⁺- 具有 dCas9 和转录共激活因子的响应性 NFAT 片段，我们利用光的力量在哺乳动物细胞中实现光诱导转录重编程。这种合成系统（称为 CaRROT）也可用于记录哺乳动物在暴露于可引发细胞内钙反应的配体或化学物质后的钙依赖性活动。