Dan Yu Lai

Short hairpin RNA (shRNA) technology enables stable and regulated gene repression. For establishing experimentally versatile RNAi tools and minimizing toxicities, synthetic shRNAs can be embedded into endogenous microRNA contexts. However, due to our incomplete understanding of microRNA biogenesis, such "shRNAmirs" often fail to trigger potent knockdown, especially when expressed from a single genomic copy. Following recent advances in design of synthetic shRNAmir stems, here we take a systematic approach to optimize the experimental miR-30 backbone. Among several favorable features, we identify a conserved element 3' of the basal stem as critically required for optimal shRNAmir processing and implement it in an optimized backbone termed "miR-E", which strongly increases mature shRNA levels and knockdown efficacy. Existing miR-30 reagents can be easily converted to miR-E, and its combination with up-to-date design rules establishes a validated and accessible platform for generating effective single-copy shRNA libraries that will facilitate the functional annotation of the genome.

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

Abstract RNA interference (RNAi) has become an indispensable tool for loss-of-function genetics across eukaryotes. By enabling stable and reversible gene silencing, short hairpin RNA (shRNA) triggers of RNAi provide a means to study long term phenotypes, perform pool-based forward genetic screens and examine the consequences of temporary target inhibition in vivo. However, efficient implementation in vertebrate systems has been hindered by our incomplete understanding of the sequence requirements for effective shRNA biogenesis and target suppression. Consequently, single-gene studies still depend on laborious testing of many candidates and pooled shRNA screens contain non-functional sequences that make the interpretation of negative results inconclusive. To address this limitation, we recently developed a high-throughput “RNAi Sensor assay” that enables functional identification of effective shRNAs at large scale. We showed that this assay allows testing of tens of thousands of candidates in parallel and robustly identifies potent single-copy shRNAs targeting virtually any gene. Here we applied this technology to evaluate 20,000 preselected candidate shRNAs targeting over 330 disease-relevant mouse genes to generate a functionally validated shRNA library for potent single-copy target knockdown. Beyond their use in RNAi transgenic animals, such shRNA libraries will enable comprehensive pooled RNAi screening and open a promising avenue for high-throughput functional genetics in the post-genomic era. Citation Format: Vaishali Sridhar, Dan Yu Lai, Nishi Sinha, Prem K. Premsrirut, Christof Fellmann. Functionally validated shRNA libraries for large-scale RNAi screens. [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 LB-319. doi:10.1158/1538-7445.AM2014-LB-319