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 complementarities. 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 function. However, owing to a tolerance for mismatches and gaps in base-pairing with targets, small RNAs could have up to hundreds of potential target sequences in a genome, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs besides their intended targets. 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). 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 transduction 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.

Vectors and parameters that enhance the efficacy of RNAi-mediated gene disruption in transgenic Drosophila

“…Overall, mismatched siRNAs are somewhat less efficient than the fully complementary siRNAs, but higher levels of the mismatched siRNAs can compensate for this reduced efficiency. These results contradict prior claims that siRNAs require at least 19 nts of pairing to induce sequence-specific gene silencing in vivo (5)(6)(7)(8).…”

Stereotypic founder cell patterning and embryonic muscle formation in Drosophila require nautilus (MyoD) gene function

“…Although off-target effects in RNAi have become a concern, nautilus dsRNA has not been reported to have offtarget issues in genomic wide screens (see http://flyrnai.org), and the similarity between the genetic and RNAi data supports this interpretation (25,26).…”

A genome-wide RNAi screen for Wnt/β-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer

“…Confounding the effective use of genome-scale RNAi screens has been the inability to discriminate between siRNAs that induce off-targeting effects or otherwise grossly affect general cellular functions, from those that target genes-of-interest (12). Directly relevant to this study is the prevalence of false positives resulting from off-targeting observed in a similar screen performed in Drosophila to identify Wnt pathway components (12,13).…”

“…Directly relevant to this study is the prevalence of false positives resulting from off-targeting observed in a similar screen performed in Drosophila to identify Wnt pathway components (12,13). To limit the influence of off-targeting in our screen, we retested 530 siRNAs of interest identified from the primary screen ( Fig.…”