Enhancing dsRNA stability and delivery in the fall armyworm, Spodoptera frugiperda

RNAi efficiency in lepidopteran insects is notoriously low. Among various factors that determine RNAi efficiency, high nuclease activity in midgut lumen of lepidopteran insects is known to be the major factor that decreases effect of dsRNA by feeding. We identified dsRNase genes in fall armyworm, endonuclease that has dsRNA degradation activity. Three dsRNase genes (dsRNase1, 1.5, and 2) were highly expressed in midgut. All three genes were in the same genomic locus. To disrupt nuclease activity in midgut, we deleted 10kb fragments which includes all three dsRNase genes coding region. We used CRISPR-Cas9 system for this deletion knockout, and sibling crossing for homozygous mutant selection. Homozygous dsRNase knockout mutant had improved dsRNA stability in midgut lumen lysate compared to wild type. However, such effect was restricted to lower lysate protein concentration. dsRNA efficiency by feeding induced slight improvement of RNAi efficiency. RNAi efficiency was further able to be improved with the help of nanoparticle by enhancing stability and delivery of the dsRNA in the dsRNase knockout strain.