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Prevalence of off-target effects in Drosophila RNA interference screens

Abstract

RNA interference (RNAi) in both plants and animals is mediated by small RNAs of approximately 21–23 nucleotides in length for regulation of target gene expression at multiple levels through partial sequence complementarities1,2. Combined with widespread genome sequencing, experimental use of RNAi has the potential to interrogate systematically all genes in a given organism with respect to a particular function3,4,5,6,7,8,9. However, owing to a tolerance for mismatches and gaps in base-pairing with targets10,11,12, small RNAs could have up to hundreds of potential target sequences in a genome13,14, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs besides their intended targets15,16. The use of long double-stranded RNAs (dsRNAs) in Drosophila, where Dicer-mediated processing produces small RNAs inside cells, has been thought to reduce the probability of such ‘off-target effects’ (OTEs)5. Here we show, however, that OTEs mediated by short homology stretches within long dsRNAs are prevalent in Drosophila. We have performed a genome-wide RNAi screen for novel components of Wingless (Wg) signal transduction17 in Drosophila S2R + cells, and found few, if any, legitimate candidates. Rather, many of the top candidates exert their effects on Wg response through OTEs on known pathway components or through promiscuous OTEs produced by tandem trinucleotide repeats present in many dsRNAs and genes. Genes containing such repeats are over-represented in candidate lists from published screens, suggesting that they represent a common class of false positives. Our results suggest simple measures to improve the reliability of genome-wide RNAi screens in Drosophila and other organisms.

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Figure 1: Candidates for positively acting Wg pathway components.
Figure 2: Suppression of Wg pathway activity by the top seven candidate dsRNAs maps to short 30–40-bp fragments.
Figure 3: Short homologies to arm account for the activity of top candidates.
Figure 4: Effects of CAN-repeat-containing dsRNAs in Wg and Hh reporter assays.

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Acknowledgements

We thank N. Perrimon and colleagues for their role in producing the dsRNA library, S. Yao for initial assistance in the Hh screen, and R. Gong for technical assistance. We also thank all members of the Beachy laboratory, R. Green and S. Dorner for helpful discussions, and G. Seydoux and J. Taipale for critical comments on the manuscript.

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Correspondence to Philip A. Beachy.

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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

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This file contains Supplementary Methods, Supplementary Figures 1–8, Supplementary Tables 1–5, and Supplementary List (Alignment between Wg screen candidates with known pathway components). (PDF 514 kb)

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Ma, Y., Creanga, A., Lum, L. et al. Prevalence of off-target effects in Drosophila RNA interference screens. Nature 443, 359–363 (2006). https://doi.org/10.1038/nature05179

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