A Toehold Switch Biosensor for Versatile miRNA Imaging and Therapeutic Application in Living Cells and Acute Liver...

Engineered biological systems are anticipated to address critical human needs in biomedical imaging, gene expression regulation, and disease theranostics. However, notable advancements in engineering genetic systems have been constrained by the limitations of the fundamental tools utilized to report, respond to, and control the intracellular components of living systems. Here, we developed a toehold switch biosensor TX-miR that extends as five specific switch systems (TF-miR-124a, TR-miR-9, TP-miR-124a, TA-miR-124a, and TN-miR-155) for in vivo imaging and therapeutics. TF-miR-124a and TR-miR-9 systems allow for the accurate monitoring of the activities of two neurospecific microRNAs (miRNA-124a, miRNA-9) during neurogenesis in living mammalian cells and mice. Moreover, the regulation of the downstream functional gene p21 enables the switch system TP-miR-124a to suppress cell proliferation by responding to target miRNAs, thereby exerting an inhibitory effect on the cell cycle. In addition, the new miRNA-induced anti-CRISPR/Cas9 system TA-miR-124a was designed by the integration of the toehold switch with the CRISPR/Cas9 system. Through the specific inhibition of Cas9 by AcrIIA4 in cells, the programmable regulation of CDK2 gene knockdown induced by the target miRNA realized cell-specific gene editing. Finally, we employed the TN-miR-155 switch to precisely modulate the expression of the Nrf-2 gene, thereby effectively mitigating the symptoms of sepsis-induced acute liver injury in mice. Together, our study demonstrates the reliability of TX-miR as a robust biosensor for versatile miRNA imaging and CRISPR/Cas9-mediated cell-type-specific gene therapeutics in disease treatment settings.