154. Applying Insights from Pri-miRNA Processing to shRNA Design

Abstract

Short hairpin RNA (shRNA) technology permits efficient and stable gene regulation, providing a useful tool for gene therapy. shRNAs can be embedded in miRNA scaffolds that allow their expression from Pol II promoters, which is more amendable to control transcription. Under this platform, shRNAs are processed through the canonical miRNA maturation pathway, which includes stepwise cleavages by Drosha-DGCR8 complex (Microprocessor) and Dicer. Despite many successes, design of Pol II driven shRNA is far from optimal. Most Pol II driven shRNAs are based on primary transcript sequence of hsa-miR-30a. Recent studies have shown that different pri-miRNAs have distinct Drosha cleavage efficiencies leading to various levels of its mature form. Here, we seek to systematically explore how different pri-miRNA scaffolds can be adapted and optimized to provide the best shRNA expression and function. By taking advantage of DGCR8 KO cells and luciferase-based reporters, we were able to directly monitor Microprocessor cleavage efficiency of primary transcripts in vivo. We have analyzed the Microprocessor cleavage of 20 different primary miRNA transcripts that are the most abundant and widely expressed. Moreover, we have explored how the preservation of stem structure and endogenous surrounding sequences and motifs contribute to shRNA maturation. This study aims to pave the way for a universal miRNA-based shRNA expression platform.