In a study published in Nature Cell Biology, the researchers from Dr. YANG Hui's team and Dr. ZHOU Haibo's team at the Institute of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology of the Chinese Academy of Sciences demonstrated that the low-abundant transcripts and long non-coding RNAs (lncRNAs) could successfully be detected. 

Detection of endogenous signals and precise control of genetic circuits in the natural context are essential to understanding biological processes. Recent studies have shown that some low-expressed genes and lncRNAs are involved in essential biological processes. The most common method for detecting the activity of endogenous genes is fluorescent protein fusion strategy, however, it was difficult to detect genes with low expression level and unable to detect lncRNAs.  

SgRNA, akin to a global positioning system (GPS), can direct a Cas nuclease to specific genome locus with high specificity, efficiency and versatility. Previous studies have reported a series of genetic switches to act as sensors or to implement additional functionality, such as inducible sgRNA to measure miRNA activity. Nevertheless, it is limited by the inability to process endogenous signals and only suitable for well-defined miRNA.  

The researchers in this study developed a broad-spectrum endogenous transcription-gated switch (Ents) that directly expresses the sgRNA precursor through an endogenous promoter. The sgRNA precursor can be processed by the endogenous release mechanism after transcription. The released sgRNA can trigger the optimized highly sensitive reporter system and induce the expression of mCherry. As a result, the activity of the endogenous transcript can be detected by SPH-OminiCMV-Ents. Notably, SPH-OminiCMV-Ents can amplify the endogenous signals and visualize the dynamics of gene expression during differentiation.  

To determine whether the SPH-OminiCMV can reliably reflect the expression level of genes, the researchers inserted the sgRNA precursor into eight genes at different expression levels including the highly expressed housekeeping gene Actb and seven pluripotency-associated genes. The results showed that the expression level of mCherry induced by SPH-OminiCMV-Ents was higher than the P2A-mCherry strategy. Besides, low-abundance genes, which are barely discernible using the P2A-mCherry strategy, can be detected via SPH-OminiCMV-Ents system. 

To improve the sensitivity of SPH-OminiCMV-Ents system, the researchers constructed a sgRNA array containing six to eight copies of the sgRNA in tandem, and each sgRNA was flanked by tRNAs. They inserted the sgRNA array at the 3'UTR of low-expressed LncRNA, enabling the visualization of LncRNA Tug1, whereas its expression could not be detected with only one copy of sgRNA. 

To identify the quantitative nature of Ents, the researchers designed a Tet-on system in which sgRNA precursor was inserted into the 3' UTR of enhanced green fluorescent protein (EGFP), and the expression level of EGFP can be controlled by the concentration of Dox. Notably, the expression level of mCherry was found to be strongly correlated with EGFP when the controllable cells were exposed to different concentrations of Dox. The result proves the accurate tracking ability of the Ents. Additionally, they also clarified the time lag between target gene and reporter at both mRNA and protein level for gene expression initiation and termination. 

This study provides a new tool to study the genetic elements in living cells. The experiments in this study were performed in culture mESCs. Future research will involve the in vivo spatio-temporal mapping and purification of defined cell populations using SPH-OminiCMV-Ents mouse model.