ABSTRACT Reverse genetic approaches in molds are complicated by their recalcitrance to transformation, multicellular growth during much of the vegetative life cycle, and frequent off-target integration of deletion constructs. Thus, protocols for gene deletion in filamentous fungi are predominantly confined to established model organisms. Adapting an existing protocol developed for gene deletion in the related fungus Aspergillus fumigatus , we employed an expression-free CRISPR/Cas9 directed mutagenesis strategy to the non-model environmental mold Aspergillus calidoustus . As a test case, we have deleted A. calidoustus pyrG , encoding orotidine-5′-phosphate decarboxylase, using short regions of homology to guide on-target integration of a nourseothricin resistance cassette (NatR) to CRISPR/Cas9-induced double strand breaks. We genotypically and phenotypically validated two A. calidoustus Δ pyrG deletion strains generated using this methodology, with whole-genome sequencing revealing one Δ pyrG strain to have integrated the resistance cassette by homologous recombination, and another strain by non-homologous end joining. Thus, distinct modes of double-strand break repair were responsible for on-target integration of the homology-bearing NatR cassette at the pyrG locus in A. calidoustus . IMPORTANCE In the environment, filamentous fungi play essential roles in soil health and agriculture, decomposition, and nutrient cycling. The study of these organisms is often limited by an inability to dissect the functions of their genes and the roles that they play by modifying the genomes of these organisms. Here, we adapt and validate a tool for genome modification to Aspergillus calidoustus , a soil fungus that forms a partnership with a bacterium ( Paraburkholderia edwinii ) to resist natural toxins found in the soil.
Hollomon et al. (2026) studied this question.