CRISPR-Cas9 mediated dsRNase knockout improves RNAi efficiency in the fall armyworm

Area of Science:

• Entomology
• Molecular Biology
• Genetics

Background:

• Lepidopteran insects exhibit resistance to RNA interference (RNAi), particularly when using orally delivered double-stranded RNA (dsRNA).
• High nuclease activity in the midgut lumen is a primary suspected cause of this RNAi insensitivity.
• The fall armyworm (FAW), Spodoptera frugiperda, is a significant agricultural pest where improving RNAi strategies is crucial.

Purpose of the Study:

• To investigate the role of midgut dsRNases in the RNAi refractory nature of lepidopteran insects.
• To develop a method for enhancing RNAi efficiency in the fall armyworm by targeting dsRNase activity.

Main Methods:

• Identification and characterization of three highly expressed dsRNase genes in the FAW midgut.
• Utilizing CRISPR-Cas9 gene editing to create a homozygous mutant FAW line with a deletion of the identified dsRNase genes.
• Assessing dsRNA degradation levels in the midgut lumen of mutant versus wild-type larvae.
• Evaluating the efficacy of RNAi targeting the inhibitor of apoptosis (IAP) gene in mutant and wild-type FAW.

Main Results:

• Three dsRNase genes were identified and found to be highly expressed in the FAW midgut.
• CRISPR-Cas9 mediated deletion of these three dsRNase genes resulted in reduced dsRNA degradation in the mutant larvae's midgut lumen.
• RNAi targeting the IAP gene led to significantly higher target gene knockdown and increased mortality in the mutant FAW larvae compared to wild-type.
• These findings demonstrate that midgut dsRNases are key contributors to RNAi inefficiency in FAW.

Conclusions:

• Midgut dsRNases play a critical role in the RNAi-deficient phenotype observed in the fall armyworm.
• Genome editing to remove dsRNase genes can enhance RNAi effectiveness in lepidopteran insects.
• Developing dsRNA formulations that protect against enzymatic degradation could improve RNAi-based pest control strategies for FAW and other lepidopterans.