RNAi experiments in D. melanogaster: solutions to the overlooked problem of off-targets shared by independent dsRNAs.
BACKGROUND
RNAi technology is widely used to downregulate specific gene products. Investigating the phenotype induced by downregulation of gene products provides essential information about the function of the specific gene of interest. When RNAi is applied in Drosophila melanogaster or Caenorhabditis elegans, often large dsRNAs are used. One of the drawbacks of RNAi technology is that unwanted gene products with sequence similarity to the gene of interest can be down regulated too. To verify the outcome of an RNAi experiment and to avoid these unwanted off-target effects, an additional non-overlapping dsRNA can be used to down-regulate the same gene. However it has never been tested whether this approach is sufficient to reduce the risk of off-targets.


METHODS
We created a novel tool to analyse the occurrence of off-target effects in Drosophila and we analyzed 99 randomly chosen genes.


RESULTS
Here we show that nearly all genes contain non-overlapping internal sequences that do show overlap in a common off-target gene.


CONCLUSIONS
Based on our in silico findings, off-target effects should not be ignored and our presented on-line tool enables the identification of two RNA interference constructs, free of overlapping off-targets, from any gene of interest.
Section of Radiation and Stress Cell Biology, Department of Cell Biology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands.
Erwin
E
Seinen
Johannes G M
JG
Burgerhof
Ritsert C
RC
Jansen
Ody C M
OC
Sibon
D000818:Animals; D001483:Base Sequence; D004331:Drosophila melanogaster / Q000235:genetics*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics*
D012330:RNA, Double-Stranded