Engineered RNAse III deletions of insect symbionts Snodgrassella alvi wkB2 and Serratia symbiotica CWBI

Symbiont-mediated RNA interference (RNAi) is a method that uses microbes that live in association with animal or plant hosts to trigger an immune response that knocks down expression of specific genes. When using double-stranded RNA (dsRNA) as the RNAi effector, degradation of dsRNA produced by engineered cells limits the effectiveness of symbiont-mediated RNAi. In this study, we deleted the RNase III gene (rnc) from the genomes of the aphid symbiont Serratia symbiotica CWBI and the honey bee symbiont Snodgrassella alvi wkB2. In other bacteria, deleting this gene has been shown to increase double-stranded RNA (dsRNA) accumulation in dsRNA-producing cells because it eliminates an enzyme that degrades dsRNA. We used a genetically encoded dsRNA sensor and other methods to validate that dsRNA levels were increased in the engineered insect symbionts. These RNase III mutant strains may be improved platforms for using symbiont-mediated RNAi to study insect biology, protect pollinator health, interrupt viral transmission by pests, and for other applications.