The growing interest for environmentally safe and species-selective pest control strategies has positioned RNA interference (RNAi) as a promising mechanism for targeted pest suppression.  An emerging strategy in this approach employs double stranded RNA (dsRNA) as the low-risk pesticide (LRP) of choice. This abstract presents dsRNA Analyzer, a computational pipeline that facilitates the design of dsRNAs with optimized specificity in a target organism (TO, i.e. a pest) and minimized off-target potential in non-target organisms (NTOs, e.g. bees, humans).

dsRNA Analyzer systematically evaluates all predicted small interfering RNAs (siRNAs) derived from an input nucleotide target sequence for their potential to elicit gene-specific knockdown. Using this information, it then proposes candidate dsRNA molecules. Customizable parameters include the desired length of the predicted siRNAs and that of the suggested dsRNA molecule and the number of allowed mismatches when screening on NTOs.

The output comprises tabulated reports that help the researcher or regulator design a dsRNA molecule with great specificity on the TO and minimal or no hits on any NTO. dsRNA Analyzer also outputs the specific patterns of any undesired siRNA hits on the NTOs providing a basis for selecting dsRNA sequences with optimal trade-offs between on-target potency and ecotoxicological safety. dsRNA Analyzer is unique in its ability to test both the genome or transcriptome of an NTO for unwanted hits.

The current implementation includes an integrated reference database of more than 90 NTO genomes and over 70 NTO transcriptomes, covering a broad range of taxonomic groups relevant to regulatory biosafety evaluations.

Future validation will experimentally assess the in silico predictions from dsRNA Analyzer’s output in Colorado potato beetle (CPB, Leptinotarsa decemlineata) as a model target organism, alongside selected NTOs representing pollinators and vertebrate species of ecological concern.

The source code of dsRNA Analyzer can be found at https://github.com/chandron/ration_v2. In addition, dsRNA Analyzer can be run as a web app at http://dsrna-analyzer.org:3000/. 

This work is supported by funding from the Horizon Europe project RATION (‘Risk assessment of low-risk pesticides’, HORIZON-CL6–2022-FARM2FORK-01, HORIZON-RIA, No. 101084163).