Institute of Translational Medicine, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, 518039, China.
Department of Otolaryngology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, 518039, China.
Nat Commun. 2017 Dec 13;8(1):2095. doi: 10.1038/s41467-017-02265-x.
The catalytically dead Cpf1 endonuclease from Acidaminococcus sp. BV3L6 (dAsCpf1) has been used to construct effective transcriptional repressors in bacteria and plants. However, it is still unclear if dAsCpf1 can function in human cells as a transcriptional regulator or a signal conductor. Here, we repurpose the dAsCpf1 system in human cells for a variety of functions, including the activation or repression of gene transcription. Moreover, we construct programmable ligand-controlled dAsCpf1 systems either by coupling crRNAs with engineered riboswitches or by fusing dAsCpf1 proteins with G protein-coupled receptors. These generalizable approaches allow us to regulate the transcription of endogenous genes in response to diverse classes of ligands, thus constructing artificial signaling pathways with rewired cellular input-output behaviors. The systems exhibit signal amplification, an important feature in cell signaling, when multiple crRNAs are processed from a single transcript. The results provide a robust and efficient platform for engineering customized cell signaling circuits.
来自 Acidaminococcus sp. BV3L6 的无催化活性的 Cpf1 内切酶（dAsCpf1）已被用于构建细菌和植物中有效的转录抑制剂。然而，目前尚不清楚 dAsCpf1 是否可以在人类细胞中作为转录调节剂或信号转导器发挥作用。在这里，我们重新设计了人类细胞中的 dAsCpf1 系统，使其具有多种功能，包括基因转录的激活或抑制。此外，我们通过将 crRNA 与工程化的核糖开关偶联，或者通过将 dAsCpf1 蛋白与 G 蛋白偶联受体融合，构建可编程配体控制的 dAsCpf1 系统。这些可推广的方法使我们能够响应各种配体来调节内源性基因的转录，从而构建具有重布线细胞输入-输出行为的人工信号通路。当从单个转录本中加工多个 crRNA 时，该系统表现出信号放大，这是细胞信号中的一个重要特征。这些结果为工程定制的细胞信号回路提供了一个强大而高效的平台。
