Building to Understand MiRNA Circuits

MicroRNA (miRNA) regulation is ubiquitous in human biology, with miRNAs playing a role in every developmental process. Despite the fact that deletion of miRNA genes typically derepress their targets by only 20%-50%, such deletions are often lethal. However, this raises the question of how such modest derepression can lead to severe phenotypic consequences. To better understand miRNA regulation, I took a "build to understand" approach: by building synthetic biological circuits using miRNA in two engineering projects, I put models of miRNA regulation to the test and created biological devices with practical applications. First, I developed miRNA incoherent feedforward loop (IFFL) circuits that enable precise control of therapeutic transgene expression to augment Rett syndrome gene therapy. Second, my colleagues and I systematically varied miRNA target complementarity and cooperativity to generate a toolkit of modular IFFL circuits, termed DIMMERs, that enabled precise, tunable control of transgene expression across diverse cell types to facilitate imaging, editing, and gene therapy. Together, these projects provided evidence that canonically-sized miRNAs can repress gene expression by more than 10-fold in the presence of three or more co-repressing miRNAs, but achieve little repression individually. This challenges previous models of miRNAs as subtle fine-tuners of gene expression, which may have underestimated miRNApotency by focusing on individual targets rather than those of cooperative groups.