Establishing a genetic part library for tunable double-stranded RNA circuit construction and RNA interference in Caenorhabditis elegans

RNA interference (RNAi), one of the major molecular tools in model nematode Ceanorhabditis elegans, relies on feeding engineered bacteria that express double-stranded RNA (dsRNA), which modulates animal host gene expression in a programmable manner. Currently no synthetic biology toolkit exits in C. elegans RNAi, while dsRNA circuits construction is restricted to one type of architecture. To facilitate systematic strategies for dsRNA circuit design and expression, here, we performed strain-specific screen of synthetic promoters, based on which we developed seventeen modular genetic parts compatible for rapid assembly of dsRNA expression constructs in Escherichia coli HT115(DE3). We validated dsRNA production in vitro and assessed RNAi efficiency in live animals by feeding. As a proof of concept, 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 establishes a synthetic biology toolkit for programmable dsRNA delivery, enabling precise control of RNAi outcomes from partial to complete gene silencing in C. elegans.