Abe Itsuki, Ohno Hirohisa, Mochizuki Megumi, Hayashi Karin, Saito Hirohide
Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, Japan.
Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan.
Nat Commun. 2025 Jul 1;16(1):5362. doi: 10.1038/s41467-025-60392-2.
RNA switch is a synthetic RNA-based technology that controls gene expression in response to cellular RNAs and proteins, thus enabling cell type-specific gene regulation and holding promise for gene therapy, regenerative medicine, and cell therapy. However, individual RNA switches often lack the specificity required for practical applications due to low ON/OFF ratios and difficulty in finding distinct and single biomolecule targets. To address these issues, we present "split RNA switches" that integrate outputs from multiple RNA switches by exploiting protein splicing. We show that split RNA switches significantly improve the ON/OFF ratio of microRNA-responsive ON switch system by canceling leaky OFF level in human cells. Using this approach, we achieve efficient cell purification using drug-resistance genes based on endogenous microRNA profiles and CRISPR-mediated genome editing with minimal off-target-cell effects. Additionally, we demonstrate RNA-based synthetic circuits using split RNA switches to enable the detection of multiple microRNAs and proteins with logical operations. Split RNA switches highlight the potential of post-translational processing as a versatile and comprehensive strategy for advancing mRNA-based therapeutic technologies.
RNA开关是一种基于合成RNA的技术，可响应细胞RNA和蛋白质来控制基因表达，从而实现细胞类型特异性的基因调控，并有望应用于基因治疗、再生医学和细胞治疗。然而，由于开/关比低以及难以找到独特的单一生物分子靶点，单个RNA开关往往缺乏实际应用所需的特异性。为了解决这些问题，我们提出了“分裂RNA开关”，它通过利用蛋白质剪接整合多个RNA开关的输出。我们表明，分裂RNA开关通过消除人类细胞中的泄漏关闭水平，显著提高了微小RNA响应开启开关系统的开/关比。使用这种方法，我们基于内源性微小RNA谱和CRISPR介导的基因组编辑，利用耐药基因实现了高效的细胞纯化，且脱靶细胞效应最小。此外，我们展示了使用分裂RNA开关的基于RNA的合成电路，以通过逻辑运算检测多种微小RNA和蛋白质。分裂RNA开关突出了翻译后加工作为推进基于mRNA的治疗技术的通用和综合策略的潜力。
