Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Schänzlestraße 18, 79104 Freiburg, Germany.
Faculty of Biology, University of Freiburg, Schänzlestraße 18, 79104 Freiburg, Germany.
Sci Adv. 2021 Jan 1;7(1). doi: 10.1126/sciadv.abd3568. Print 2021 Jan.
Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.
光诱导基因开关代表了一种在基础和应用研究中精确操纵细胞事件的关键策略。然而，由于光刺激的组织穿透力低和可能的光毒性，广泛使用的基因开关的性能受到限制。为了克服这些限制，我们设计了光遗传学合成转录因子，使其在与启动子紧密空间接近的情况下发生液-液相分离。通过引入无序区域来实现组成型和光遗传学合成转录因子的相分离。在定量数学模型的支持下，我们证明了工程化转录因子液滴在靶启动子处形成，并将基因表达提高了五倍。这种性能的提高在多种哺乳动物细胞系以及原位转染后的小鼠中都得到了观察。这项工作的结果表明，引入无规卷曲结构域是一种简单而有效的方法，可以提高合成转录因子的活性。
