Short hairpin RNA (shRNA) mediated RNAi is a well-established method for investigating cancer pathways and is a promising future therapeutic strategy. In particular, delivery of shRNAs by retroviral transduction enables RNAi knockdown in mouse models and cell lines refractory to transfection. The primary disadvantage of retroviral shRNA-based experiments is the reduced target gene knockdown frequently observed when a single copy of the shRNA vector is integrated. We have investigated the fate of shRNA precursors using a novel deep sequencing strategy we have previously developed. We find over 99 percent of precursor molecules are diverted to a non-productive biogenesis route, severely limiting the amount of mature RNA produced. In this proposed application we describe a screening strategy to optimize shRNA design and maximize target gene knockdown potential. Our long term goal, beyond the scope of this grant application, is to construct genome-wide shRNA libraries using our optimized shRNA design.
RNAi gene silencing tools have found widespread use in the study of cancer pathways. The major limitation of RNAi technology is the incomplete level of gene silencing. This project will screen for the optimal design to maximize the efficiency of gene silencing.
