Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China.
Nat Cell Biol. 2021 Jan;23(1):99-108. doi: 10.1038/s41556-020-00610-9. Epub 2021 Jan 4.
Detection of endogenous signals and precise control of genetic circuits in the natural context are essential to understand biological processes. However, the tools to process endogenous information are limited. Here we developed a generalizable endogenous transcription-gated switch that releases single-guide RNAs in the presence of an endogenous promoter. When the endogenous transcription-gated switch is coupled with the highly sensitive CRISPR-activator-associated reporter we developed, we can reliably detect the activity of endogenous genes, including genes with very low expression (<0.001 relative to Gapdh; quantitative-PCR analysis). Notably, we could also monitor the transcriptional activity of typically long non-coding RNAs expressed at low levels in living cells using this approach. Together, our method provides a powerful platform to sense the activity of endogenous genetic elements underlying cellular functions.
在自然环境中检测内源性信号和精确控制遗传回路对于理解生物过程至关重要。然而，处理内源性信息的工具是有限的。在这里，我们开发了一种可推广的内源性转录门控开关，在存在内源性启动子时释放单链向导 RNA。当内源性转录门控开关与我们开发的高灵敏度 CRISPR 激活物相关报告基因偶联时，我们可以可靠地检测内源性基因的活性，包括表达水平非常低的基因（相对于 Gapdh，定量 PCR 分析<0.001）。值得注意的是，我们还可以使用这种方法在活细胞中监测通常低水平表达的长非编码 RNA 的转录活性。总之，我们的方法为感知细胞功能的内源性遗传元件的活性提供了一个强大的平台。
