Xu, M.; Ireri, S. W.; Prator, M.; Lostroh, P.; Cao, M.

Bacteria can be engineered to express double-stranded RNA (dsRNA) that modulates eukaryotic host gene expression in a programmable manner via RNA interference (RNAi). This requires robust and systematic strategies for dsRNA circuit design and expression. Here, we developed modular genetic parts compatible with the CIDAR MoClo system for rapid assembly of dsRNA expression constructs in Escherichia coli HT115(DE3). We validated dsRNA production in vitro and assessed RNAi efficiency in Caenorhabditis elegans. A constitutive dsRNA circuit achieved rapid and near-complete gene knockdown, whereas a Ptac-driven circuit enabled tunable, partial silencing while minimizing the leakiness commonly observed in standard feeding RNAi systems. Together, this work expands the synthetic biology toolkit for dsRNA delivery, enabling precise control of RNAi outcomes from partial to complete gene silencing in nematodes.