Co-transformation of Aspergillus fumigatus: a simple and efficient strategy for gene editing without linking selectable markers

Understanding the basis of fungal pathogenesis requires knowledge of pathogen biology that is built through studies of gene function and regulation. The critical first step in nearly all these studies is genetic transformation: the generation of targeted DNA sequence modifications through the introduction of exogenous DNA into the cell. For research focused on gene regulation, or where small precise mutations are desired, the maintenance of genomic context (i.e. surrounding DNA sequences) is important as the disruption of flanking DNA elements can alter gene expression and confound results. This often makes the inclusion of selectable markers that are physically linked to the sequence of interest unsuitable and complicates the transformation process.   Here, we present a co-transformation strategy in the human pathogen Aspergillus fumigatus that can be used to make precise, marker-free gene edits at a locus of interest without disturbing flanking DNA sequences. By simultaneously introducing a marker-free, modified copy of the gene of interest and a plasmid that directs the integration of a selectable marker to a different locus, this approach takes advantage of the benefits of selection, with results similar to that of a truly marker-less strategy.