Abstract LB-319: Functionally validated shRNA libraries for large-scale RNAi screens

Abstract

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