Developing a Highly Flexible, Inducible, and Trackable microRNA-Based System for Perturbing Gene Expression

The ability to reliably and selectively knock down genes in mammalian cells opens up numerous possibilities for medical and research applications, such as disease modeling and cancer therapeutics. RNA interference is an integral tool used for gene knockdown that facilitates study of the effects of genes in complex endogenous networks. One class of RNA used, short hairpin RNA (shRNA), acts as a post-transcriptional gene regulator through sequence-specific transcript degradation and/or translational repression. Traditional shRNA expression methods provide no control or measure of shRNA expression after delivery. It would be desirable for researchers to have tools to selectively induce knockdown against a target of choice through conditional shRNA expression and to more easily and reliably track cells that are correctly producing these shRNAs. Synthetic microRNA scaffolds that contain shRNA sequences, known as shRNAmirs, enable conditional expression with small molecule-based induction or cell type-specific promoters that is not achievable with traditional shRNA expression methods. Here, we develop a robust, customizable, and inducible platform for gene knockdown by embedding an intronic shRNAmir between exons of the SNAP protein tag, which can be covalently labeled by a fluorescent dye of choice. This design couples expression of shRNAmirs with the splicing of SNAP to provide a fluorescent readout of processed shRNA levels. We show that intronic shRNAmirs targeting sequences in exons or UTRs lead to reduced target gene levels, and greater knockdown is correlated with higher SNAP expression. We then put this gene construct under the control of a doxycycline-inducible promoter, providing temporal control of shRNA expression. We also show in mammalian cells that SNAP shRNAmirs can knock down reporters tagged with previously published shRNA target sequences at levels close to those of traditionally expressed shRNAs, highlighting the effectiveness and flexibility of the platform. Overall, coupling shRNAmir expression to SNAP splicing in a small molecule-inducible system provides a convenient way to selectively express shRNA against targets of choice and simultaneously track expression levels with a customizable output signal. This system will help researchers working with shRNA-based knockdown methods more easily understand effects of knockdown on their gene expression systems.