TY - JOUR T1 - Expression of short hairpin RNAs using the compact architecture of retroviral microRNA genes JF - bioRxiv DO - 10.1101/131581 SP - 131581 AU - James M. Burke AU - Rodney P. Kincaid AU - Francesca Aloisio AU - Nicole Welch AU - Christopher S. Sullivan Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/04/27/131581.abstract N2 - Short hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, the U6 and H1 promoters encompass the majority of the ∼270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by greater than two-fold as compared to the U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting. ER -