Knockout of SldsRNase1 and SldsRNase2 revealed their function in dsRNA degradation and contribution to RNAi efficiency in the tobacco cutworm, Spodoptera litura

Abstract

The application of RNA interference (RNAi) methods to control lepidopteran pests is difficult because of lower RNAi efficiency in these insects. Numerous studies concluded that dsRNA degrading nucleases (dsRNases) could be one of the factors involved in reducing RNAi efficiency in insects. The knockdown of genes coding for dsRNases is not successful due to inefficient RNAi in these insects. CRISPR/Cas9 system could help to knockout genes coding for dsRNases. Here, we knocked out two gut-specific dsRNase genes identified in the previous studies by using CRISPR/Cas9 system. The homozygous mutant strains containing knockout of one of the two dsRNases (SL1KO or SL2KO) and the double genes homozygous strain with knockout of both dsRNases (SL1SL2KO) showed 79.3%, 40.4% and 96.3% decrease in dsRNA-degrading activity, respectively. The nuclease activity in the gut fluid and the hemolymph was decreased by 83.2% and 23.3%, respectively, in SL1SL2KO strain as compared to the WT. Also, the level of fed dsRNA was almost 20 times higher in the SL1SL2KO strain than that in WT strain, but a similar dsRNA persistence time as detected in the hemolymph collected from SL1SL2KO and WT. Comparative analysis of RNAi response between SL1SL2KO and the WT strain showed that knockout of both dsRNases increased RNAi efficiency as the target gene mRNA levels were suppressed by feeding dsRNA to SL1SL2KO.