Knockout of chitin synthase gene confers resistance to Bt toxin Vip3Aa in Helicoverpa zea

Authors: Heu, Chan; SCHUTZE, INANA - Oak Ridge Institute For Science And Education (ORISE); Leroy, Dannialle; WANG, YU-HUI - Oak Ridge Institute For Science And Education (ORISE); DEGAIN, BEN - University Of Arizona; KERNS, DAWSON - University Of Tennessee; ABDELGAFFAR, HEBA - University Of Tennessee; JURAT-FUENTES, JUAN LUIS - University Of Tennessee; MATZKIN, LUCIANO - University Of Arizona; CARRIERE, YVES - University Of Arizona; TABASHNIK, BRUCE - University Of Arizona; Fabrick, Jeffrey

Submitted to: Pest Management Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/8/2025
Publication Date: 9/21/2025
Citation: Heu, C.C., Schutze, I.X., LeRoy, D.M., Wang, Y., DeGain, B.A., Kerns, D.D., Abdelgaffar, H., Jurat-Fuentes, J., Matzkin, L.M., Carriere, Y., Tabashnik, B.E., Fabrick, J.A. 2025. Knockout of chitin synthase gene confers resistance to Bt toxin Vip3Aa in Helicoverpa zea. Pest Management Science. 82(1):911-919. https://doi.org/10.1002/ps.70248.
DOI: https://doi.org/10.1002/ps.70248

Interpretive Summary: Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) are used globally to manage key insect pests. However, resistance to Bt proteins in at least 11 pest species has reduced the effectiveness of Bt crops, leading to economic losses for U.S. farmers. Helicoverpa zea (also known as bollworm and corn earworm), one of the most economically damaging pests in the United States, has evolved resistance to the crystalline (Cry) Bt proteins and early warning of resistance to the vegetative insecticidal protein Vip3Aa has been reported. Here, an ARS scientist at Maricopa, Arizona, and collaborators used CRISPR/Cas9 gene editing to knock out a gene that is associated with Vip3Aa resistance in other insect pests. The H. zea knockout strain had 29,000-fold resistance to Vip3Aa which was inherited as a recessive trait and did not cause cross-resistance to other Cry toxins produced in Bt corn and cotton. Results increase our understanding of the genetic basis of H. zea resistance to Vip3Aa which is needed to monitor, manage, and counter resistance, thereby preserving the Bt technology as an effective pest management tool for U.S. cotton and corn producers.

Technical Abstract: BACKGROUND: Genetically engineered crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are used globally to manage key insect pests, including Helicoverpa zea (Lepidoptera: Noctuidae), a major pest of crops in the Americas. However, pests of at least 11 species, including H. zea, have evolved resistance to Bt crops, diminishing their effectiveness and benefits. For H. zea in the United States, practical resistance to Bt corn and cotton producing crystalline (Cry) Bt proteins is widespread and early warning of resistance to the vegetative insecticidal protein Vip3Aa has been reported. Thus, a better understanding of the genetic basis of resistance to Vip3Aa is needed to monitor, manage, and counter resistance. In some strains of lepidopteran pests, resistance to Vip3Aa is associated with disruptive mutations in the chitin synthase 2 (CHS2) gene but this association had not been investigated previously in H. zea. RESULTS: Here, we show that mutations introduced by CRISPR/Cas9 editing of the CHS2 gene can cause resistance to Vip3A in H. zea. Disruptive mutations in CHS2 facilitated the creation of strain CHS2-KO that had 29,000-fold resistance to Vip3Aa relative to its unedited parental susceptible strain. Resistance to Vip3Aa in CHS2-KO was autosomal, recessive, and did not cause cross-resistance to Cry1Ac or Cry2Ab. CONCLUSION: Results of this study indicate that CHS2 plays an important role in Vip3Aa intoxication in H. zea. It will be important to determine if mutations in CHS2 contribute to field-evolved resistance to Vip3Aa in H. zea and other pests.