Building RNA-Mediated Artificial Signaling Pathways between Endogenous Genes

Sophisticated genetic networks play a pivotal role in orchestrating cellular responses through intricate signaling pathways across diverse environmental conditions. Beyond the inherent complexity of natural cellular signaling networks, the construction of artificial signaling pathways (ASPs) introduces a vast array of possibilities for reshaping cellular responses, enabling programmable control of living organisms. ASPs can be integrated with existing cellular networks and redirect output responses as desired, allowing seamless communication and coordination with other cellular processes, thereby achieving designable transduction within cells. Among diversified ASPs, establishing connections between originally independent endogenous genes is of particular significance in modifying the genetic networks, so that cells can be endowed with new capabilities to sense and deal with abnormal factors related to differentiated gene expression (i.e., solve the issues of the aberrant gene expression induced by either external or internal stimuli). In a typical scenario, the two genes X and Y in the cell are originally expressed independently. After the introduction of an ASP, changes in the expression of gene X may exert a designed impact on gene Y, subsequently inducing the cellular response related to gene Y. If X represents a disease signal and Y serves as a therapeutic module, the introduction of the ASP empowers cells with a new spontaneous defense system to handle potential risks, which holds great potential for both fundamental and translational studies.In this Account, we primarily review our endeavors in the construction of RNA-mediated ASPs between endogenous genes that can respond to differentiated RNA expression. In contrast to other molecules that may be restricted to specific pathways, synthetic RNA circuits can be easily utilized and expanded as a general platform for constructing ASPs with a high degree of programmability and tunability for diversified functionalities through predictable Watson-Crick base pairing. We first provide an overview of recent advancements in RNA-based genetic circuits, encompassing but not limited to utilization of RNA toehold switches, siRNA and CRISPR systems. Despite notable progress, most reported RNA circuits have to contain at least one exogenous RNA X as input or one engineered RNA Y as a target, which is not suitable for establishing endogenous gene connections. While exogenous RNAs can be engineered and controlled as desired, constructing a general and efficient platform for manipulation of naturally occurring RNAs poses a formidable challenge, especially for the mammalian system. With a focus on this goal, we are devoted to developing efficient strategies to manipulate cell responses by establishing RNA-mediated ASPs between endogenous genes, particularly in mammalian cells. Our step-by-step progress in engineering customized cell signaling circuits, from bacterial cells to mammalian cells, from gene expression regulation to phenotype control, and from small RNA to long mRNA of low abundance and more complex secondary structures, is systematically described. Finally, future perspectives and potential applications of these RNA-mediated ASPs between endogenous genes are also discussed.