A sequence complementarity-based approach for evaluating off-target transcript knockdown in Bombus terrestris , following ingestion of pest-specific dsRNA

Double-stranded RNA (dsRNA) designed to target genes in a sequence-specific manner can be valuable in pest control. However, in scenarios where a nontarget could be susceptible and is predicted to be exposed to insecticidal dsRNA in the environment, it is useful to understand possible gene silencing effects in the nontarget. In this study, a sequence complementarity-based approach was used to evaluate potential gene silencing effects in the bumblebee, Bombus terrestris , following oral exposure to a fusion dsRNA product ( Ba -dsCOP-vg) specifically designed to target both alpha-coatomer ( alphaCOP ) and vitellogenin ( vg ) in the pest pollen beetle, Brassicogethes aeneus . The susceptibility of bumblebees to ingested dsRNA was first evaluated by feeding them with bumblebee-specific dsRNA ( Bt -dsCOP-vg) (1 µg/µl) targeting its alphaCOP and vg . A respective 50% and 47% reduction in the transcripts of alphaCOP and vg at 96 h post exposure confirmed its susceptibility to the ingested dsRNA. Using computational tools, a pool of all possible continuous 22-nucleotide stretches from Ba- dsCOP-vg was generated and mapped to all potential off-target genes in the B. terrestris genome, with at least a 13-nt match within the 22-nt stretch . A set of criteria based on sequence complementarity was then used to select 24 potential off-targets for transcript analysis in bumblebees. Following ingestion of Ba- dsCOP-vg (1 µg/µl), no reduction was found in the transcript level for all off-targets, including an off-target with 20-continuous-nt matches. Our findings provide guidance to future risk analysis of dsRNA products and for risk assessments frameworks that incorporate sequence complementarity-based analysis for off-target predictions.