SIREN: Suite for Intelligent RNAi Design and Evaluation of Nucleotide Sequences

Motivation RNA interference (RNAi) is a powerful tool for gene silencing across biological research, therapeutics, and agriculture. While siRNA design has benefited from advances in thermodynamic modeling and machine learning, comprehensive tools for designing long double-stranded RNAs (dsRNAs) with minimized off-target effects remain limited. Results Here, we present SIREN, an open-source Python pipeline designed to streamline RNAi construct design. SIREN integrates siRNA generation, thermodynamically-informed off-target prediction, scoring of dsRNA candidates based on cumulative off-target effects, and primer design for in vitro synthesis. It accepts user-defined transcriptomes for context-specific analysis and provides adjustable sensitivity settings balancing accuracy and computational demands. Benchmarking with plant, oomycete, and human transcriptomes demonstrates SIREN’s efficient scalability and the practical utility of medium sensitivity, recovering over 75% of high-sensitivity targets with significantly reduced computing times. Experimental validation in Phytophthora capsici confirms that SIREN effectively identifies highly specific RNAi constructs with no detectable off-target phenotypes in host plants. Availability and implementation SIREN is implemented in Python 3 and available under an open-source license at https://github.com/pablovargasmejia/SIREN ; installer via PyPI: https://pypi.org/project/siren-rnai/ .