Knowledge of the genetics and mechanisms of pest resistance is urgently needed to assess and manage the risk of resistance for widely adopted transgenic Bt crops, yet this information is lacking for Helicoverpa zea (corn earworm, CEW), one of the most economically important crop pests in the United States. Some populations of this pest have evolved resistance to Cry1 and Cry2 toxins currently deployed in Bt crops, and Vip3Aa remains the only Bt toxin in multi-toxin Bt corn and cotton that is highly effective against populations of this pest in several regions.To improve knowledge on pest resistance to crops producing Vip3Aa, this project will use a genome wide association study, high-resolution mapping, RNA sequencing, and CRISPR/Cas9 gene editing to identify genes conferring resistance to Vip3Aa in CEW. By developing genomic tools for molecular detection of Vip3Aa resistance in CEW, we will enhance resistance risk assessment and management for CEW and provide new tools for preserving efficacy of Bt crops in the U.S. Specifically, we will identify genes conferring resistance to Vip3Aa in lab- and field-selected populations of CEW and compare the resistance genes between the lab- and field-selected populations. We will confirm the role of identified genes by using CRISPR/Cas9 gene editing to create knockout mutations in a Vip3Aa-susceptible strain that disrupt candidate resistance genes for Vip3Aa. The new knowledge on genes conferring resistance to Vip3Aa will be used to track the frequency of alleles conferring resistance to Vip3Aa over four years across CEW populations from five states (AR, LA, MS, TN and TX) where the resistance risk is high.This project will facilitate regulator's decisions in response to evolution of pest resistance to Bt crops. This research should contribute in maintaining the durability of Bt traits and thus benefit growers and seed providers. The novel techniques developed and fundamental knowledge gained in this project may have immediate and broad applications for monitoring and managing resistance in other systems, and thus benefit other scientists/researchers. We anticipate that two post-docs (one individual for one year; the other for several months) and several under graduate students (two per year, 10 h per week) will be involved in this research and receive training in entomology and molecular aspects of Bt resistance.