Identification and Purification of Specific Cell Populations via ADAR Editing-Driven Synthetic Genetic Circuits

Cell separation and purification techniques are crucial in modern biomedical research and clinical applications. Endogenous RNA, which reflects a cell's genetic and physiological characteristics, provides a new way to determine cell identity at the transcriptional level. Here, we utilize RNA editing technology based on adenosine deaminase acting on RNA (ADAR) to design a dual-switch genetic circuit capable of detecting unique RNA biomarkers for cell separation and purification. The circuit incorporates a kill switch driven by barnase, which selectively eliminates nontarget cells, and a recognition switch, precisely regulated by ADAR editing, to control the expression of the MS2 bacteriophage coat protein (MCP) and barstar that inhibit barnase expression and activity. By temporally regulating these switches, our approach achieves purification efficiencies of 93-97% for HepG2, A549, and HER2-overexpressing SK-BR-3 cells in mixed populations, surpassing traditional methods. Furthermore, utilizing standard cell culture protocols, our approach simplifies cell identification and purification without interfering with the normal gene expression of target cells, ensuring robustness and safety. We believe that this ADAR-assisted genetic circuit holds great potential for applications in cell therapy and biopharmaceutical manufacturing.