A microRNA-gated thgRNA platform for multiplexed activation of gene expression in mammalian cells

To effectively reprogram cellular regulatory networks towards desired phenotypes, it is critical to have the ability to provide precise gene regulation in a spatiotemporal manner. We have previously engineered toehold-gated guide RNA (thgRNA) to enable conditional activation of dCas9-mediated transcriptional upregulation in mammalian cells using synthetic RNA triggers. Here, we demonstrate that microRNA (miR)-gated thgRNAs can be transcribed by type II RNA polymerase to allow multiplexed transcriptional activation using both mRNA and miR. Activation is achieved only by proper miR-mediated processing of the flanking 5' cap and 3' poly A tail and hairpin unblocking by mRNA via strand displacement. This new AND-gate design is exploited to elicit conditional protein degradation based on induced expression of a specific ubiquibody. This new strategy may find many new applications in an RNA-responsive manner.