Non-reversible atoxigenic aspergillus strains for aflatoxin management

Overview

Aflatoxin contamination poses a significant threat to the safety and marketability of staple crops including peanut, corn, and cotton. Existing competitive atoxigenic fungal technology (CAFT), while EPA-approved and successful in the US and Kenya, carries an inherent risk: soil sampling has revealed that atoxigenic strains can potentially revert to toxigenic strains through mating with compatible wild-type strains. This research addresses that critical limitation by developing non-reversible atoxigenic Aspergillus strains using CRISPR-mediated gene drive technology. By targeting both the vital aflatoxin production gene AflC and the mating type gene MAT1-2, the resulting strains can displace toxigenic populations while remaining permanently atoxigenic, even in the presence of compatible mating partners.

Technical specifications

Key features:

• CRISPR-Cas9 construct with four expression cassettes targeting the AflC aflatoxin biosynthesis gene and the MAT1-2 mating type gene
• Gene drive mechanism ensures the atoxigenic trait propagates to mating-compatible wild-type strains
• Insertional mutagenesis of AflC prevents aflatoxin production while preserving strain viability and competitiveness
• Disruption of MAT1-2 blocks sexual reproduction with compatible toxigenic strains, preventing reversion
• Optimized PEG-mediated protoplast transformation protocol for A. flavus
• Hygromycin selection marker for reliable transformant identification
• Validation through DNA sequencing, ELISA, LC-MS, and dual culture displacement assays

Technology readiness level

The technology is currently at laboratory proof-of-concept stage. CRISPR-mediated gene drives have been validated in yeast, Anopheles mosquitoes, and fruit fly, and natural AflC mutants have demonstrated atoxigenicity and displacement capability in Aspergillus. The research team has already developed the CRISPR construct and optimized transformation procedures. Remaining validation steps over the next year include transforming the aflatoxigenic A. flavus MAT1-1 strain, confirming genome-editing machinery integration by sequencing, testing aflatoxin production via ELISA and LC-MS on infected peanut seeds, evaluating displacement of toxigenic strains in dual culture, and confirming MAT1-2 mutagenesis in co-culture. Successful completion will advance the technology toward field trials and eventual regulatory approval for agricultural deployment.