Tan, X. et al. Proc. Natl. Acad. Sci. USA 109, 869–874 (2012).

RNA interference is an effective way to knock down gene expression, but the secondary structure and mutability of viral RNA genomes make for challenging targets. Tan et al. applied a high-throughput screening system for silencing short hairpin RNA (shRNA) to all possible target sites of hepatitis C, H1N1 influenza A, and two human immunodeficiency virus genomes. They used over 40,000 barcoded probes to make a library of sensors with a targeting shRNA under inducible control and a target site–bearing fluorescent reporter to report knockdown activity. Screening the pooled library in cells by fluorescence-activated cell sorting and microarray hybridization allowed identification of highly effective shRNAs. Potency was dependent on distinct target sequence features, secondary structure and G+C content.