Creating large chromosomal segment deletions in Aspergillus flavus by a dual CRISPR/Cas9 system: Deletion of gene clusters for production of aflatoxin, cyclopiazonic acid, and ustiloxin B

Authors: Chang, Perng Kuang

Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/26/2023
Publication Date: 12/27/2023
Citation: Chang, P.-K. 2023. Creating large chromosomal segment deletions in Aspergillus flavus by a dual CRISPR/Cas9 system: Deletion of gene clusters for production of aflatoxin, cyclopiazonic acid, and ustiloxin B. Fungal Genetics and Biology. 170:103863. https://doi.org/10.1016/j.fgb.2023.103863.
DOI: https://doi.org/10.1016/j.fgb.2023.103863

Interpretive Summary: Preharvest aflatoxin contamination of crops by aflatoxigenic Aspergillus flavus is a serious problem. To prevent A. flavus colonization and subsequent aflatoxin contamination, biological control, which employs non-aflatoxigenic A. flavus strains to outcompete toxigenic A. flavus populations, is currently the most cost-effective technology that has demonstrated the greatest efficacy. A concern regarding the potential of component strains in current biocontrol products to produce other mycotoxins has recently been raised. In this study, a genetic engineering method that can efficiently remove large chromosomal DNA segments from A. flavus genome as evidenced by deletions of three gene clusters involved in production of toxic metabolites was developed The deletion protocol is purely a subtraction technique, and no foreign DNA is integrated into genomes of derived atoxigenic A. flavus strains. It could facilitate the approval of applications for experimental use permits by the US Environmental Protection Agency.

Technical Abstract: Aspergillus flavus produces hepatocarcinogenic aflatoxin that adversely impacts human and animal health and international trade. A promising means to manage preharvest aflatoxin contamination of crops is biological control, which employs non-aflatoxigenic A. flavus isolates possessing defective aflatoxin gene clusters to outcompete field toxigenic populations. However, these isolates often produce other toxic metabolites. The CRISPR/Cas9 technology has greatly advanced genome editing and gene functional studies. Its use in deleting large chromosomal segments of filamentous fungi is rarely reported. A system of dual CRISPR/Cas9 combined with a 60-nucleotide donor DNA that allowed complete removal of three A. flavus gene clusters involved in production of harmful secondary metabolites was established. It efficiently deleted a 102-kb segment containing both aflatoxin and cyclopiazonic acid gene clusters from toxigenic A. flavus morphotypes, L-type and S-type. It further deleted the 27-kb ustiloxin B gene cluster of a resulting L-type mutant. Overall efficiencies of deletion ranged from 66.6% to 85.6%. To determine the capacity of this technique, a pigment-screening setup based on absence of aspergillic acid gene cluster was devised. Chromosomal segments of 201 kb and 301 kb were deleted with efficiencies of 57.7% to 69.2%, respectively. This system used natural A. flavus isolates as recipients, eliminated a forced-recycling step for producing recipients for next round deletion, and generated maker-free deletants with sequences predefined by donor DNA. The research provides a method for creating genuine atoxigenic biocontrol strains friendly for field trial release. It is also applicable to producing improved strains of food-grade Aspergillus oryzae and Aspergillus sojae.