ABSTRACTRNA interference (RNAi) offers an efficient way to repress genes of interest, and it is widely used in research settings. Clinical applications emerged more recently, with 5 approved siRNAs (the RNA guides of the RNAi effector complex) against human diseases. The development of siRNAs against the SARS-CoV-2 virus could therefore provide the basis of novel COVID-19 treatments, while being easily adaptable to future variants or to other, unrelated viruses. Because the biochemistry of RNAi is very precisely described, it is now possible to design siRNAs with high predicted activity and specificity using only computational tools. While previous siRNA design algorithms tended to rely on simplistic strategies (raising fully complementary siRNAs against targets of interest), our approach uses the most up-to-date mechanistic description of RNAi to allow mismatches at tolerable positions and to force them at beneficial positions, while optimizing siRNA duplex asymmetry. Our pipeline proposes 8 siRNAs against SARS-CoV-2, andex vivoassessment confirms the high antiviral activity of 6 out of 8 siRNAs, also achieving excellent variant coverage (with several 3-siRNA combinations recognizing each correctly-sequenced variant as of September2022). Our approach is easily generalizable to other viruses as long as avariant genome database is available. With siRNA delivery procedures being currently improved, RNAi could therefore become an efficient and versatile antiviral therapeutic strategy.