Abstract 4273: An enhanced microRNA backbone for potent single-copy RNAi

Abstract

Abstract Short-hairpin RNA (shRNA) technology enables stable and regulated gene repression through RNA interference (RNAi). Synthetic shRNAs can be embedded into the context of an endogenous microRNA (miRNA) to create natural substrates of RNAi pathways and minimize cellular toxicities. While such “shRNAmir” reagents provide an experimentally versatile RNAi system, their design remains challenging due to our limited understanding of miRNA processing factors. Consequently, shRNAmir reagents often fail to trigger potent target knockdown, especially when expressed from a single genomic integration (“single-copy”), a common scenario in pool-based screens and other key applications. To tackle this issue, we recently developed a high-throughput Sensor assay that enables functional identification of effective shRNAs and has been used to establish shRNA-specific prediction rules. Following these advances in design of the synthetic shRNA stem, here we take a systematic approach to improve the experimental miR-30 backbone. Among several sequence features enhancing knockdown efficiency, we identify a conserved element 3′ of the basal stem that is critically required for optimal shRNAmir processing. Implementation of this feature in an optimized backbone termed “miR-E” results in a 10-30 fold increase in mature shRNA levels, and generally boosts knockdown efficacy. Importantly, combination of the enhanced miR-E backbone with Sensor-based design rules establishes a validated and easily accessible platform for generating single-copy effective shRNAs for pool-based RNAi screening and transgenic animals, and will facilitate the functional annotation of the genome. Citation Format: Christof Fellmann, Thomas Hoffmann, Vaishali Sridhar, Barbara Hopfgartner, Dan Yu Lai, Johannes Zuber. An enhanced microRNA backbone for potent single-copy RNAi. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4273. doi:10.1158/1538-7445.AM2014-4273