Design and evaluation of genome-wide libraries for RNAi screens

This peer-reviewed article was published immediately upon acceptance. It can be downloaded, printed and distributed freely for any purposes (see copyright notice below). which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT RNA interference screens have enabled the systematic analysis of many biological processes in cultured cells and whole organisms. The success of such screens and the interpretation of the data depend on the stringent design of RNAi libraries. We describe and validate NEXT-RNAi, a software for the automated design and evaluation of RNAi sequences on a genome-wide scale. NEXT-RNAi is implemented as open-source software and is accessible at 3 RATIONALE RNA interference (RNAi) screens have become an important tool for the identification and characterization of gene function on a large-scale and complement classic mutagenesis screens by providing a means to target almost every transcript in a sequenced and annotated genome. RNAi is a post-transcriptional gene silencing mechanism conserved from plants to humans and relies on the delivery of exogenous short double-stranded (ds) RNAs that trigger the degradation of homologous mRNAs in cells [1, 2]. As an experimental tool, RNAi is now widely used to silence the expression of genes in a broad spectrum of organisms [3]. The availability of genome-wide RNAi libraries for cell-based assays and whole organisms has opened new avenues to query genomes for a broad spectrum of loss-of-function phenotypes [4, 5]. The number of sequenced genomes is steadily rising, enabling reverse genetic approaches using RNAi in many novel model systems, including e.g. the medically relevant vector Anopheles gambiae and species used to study evolutionarily aspects of development such as Tribolium castaneum, Acyrthosiphon pisum and Schmidtea mediterranea. RNAi libraries will facilitate the functional characterization of genes in these species, either through studying smaller subsets of candidates or on a genomic scale. The design of RNAi reagents is key for obtaining reliable phenotypic data in large-scale RNAi experiments. Several recent studies demonstrated that the degradation of non-intended transcripts (so-called " off-target effects ") and knock-down efficiency depend on the sequence of the RNAi reagent and have to be carefully monitored [6-13]. Based on experimental studies, rules for the design of RNAi 4 reagents have been devised to improve knock-down efficiency and simultaneously minimize unspecific effects. In invertebrates such as C. elegans and Drosophila RNAi can be triggered by long dsRNAs that are intracellularly broken down into short interfering (si) …