Embedding middle-scale artificial gene networks in live mammalian cells is one of the most important future goals for cell engineering. However, the applications of the highly orthogonal and conventional artificial transcription factors currently available are limited. In this study, we present a scalable pipeline to produce artificial transcription factors based on homing endonucleases, also known as meganucleases. The introduction of mutations at critical sites for nuclease activity renders these homing endonucleases a simple but highly specific DNA binding domain for their specific DNA target. The introduction of inactivated meganucleases linked to transcriptional activator domains strongly induced reporter gene expression, while their fusion to transcriptional repressor domains suppressed them. In addition, we show that inactivated meganuclease-based transcription factors could be embedded in the synthetic membrane receptor synNotch and used to construct synthetic circuits. These results suggest that inactivated meganucleases are useful DNA-binding domains for the construction of synthetic transcription factors in mammalian cells.

中文翻译：

---

基于大范围核酸酶的人工转录因子。

在活的哺乳动物细胞中嵌入中等规模的人工基因网络是细胞工程最重要的未来目标之一。然而，目前可用的高度正交和常规人工转录因子的应用是有限的。在这项研究中，我们提出了一个可扩展的管道来生产基于归巢核酸内切酶（也称为大范围核酸酶）的人工转录因子。在核酸酶活性的关键位点引入突变使这些归巢核酸内切酶成为其特定 DNA 靶标的简单但高度特异性的 DNA 结合域。与转录激活子域相关联的灭活大范围核酸酶的引入强烈诱导报告基因表达，而它们与转录抑制域的融合抑制了它们。此外，我们表明失活的基于大范围核酸酶的转录因子可以嵌入合成膜受体 synNotch 并用于构建合成电路。这些结果表明灭活的大范围核酸酶是有用的 DNA 结合域，可用于在哺乳动物细胞中构建合成转录因子。