Providing a toolbox for genomic engineering of .

Fungi belonging to the genus of have a long history of infecting crops of edible fungi and reducing the yield. is the main causal agent of green mold disease in . Despite its infamous role as a persistent and aggressive contamination in crops, can also be used as a biocontrol agent or as a promoter of plant growth. In order to work efficiently with on a molecular biology level, a transformation protocol is required. This study provides a detailed protocol on how to perform a transformation in using plasmid DNA for ectopic integration. In addition, a Cas9-RNP-based approach has been established for genome editing. We performed two transformations to confirm the usability of the gene as well as the gene from as selection markers. First, we integrated the gene ectopically and determined the overall transformation efficiency. In the second transformation experiment, we disrupted the ornithine-5' phosphate decarboxylase gene of by using the CRISPR-Cas9 approach. In total, four candidates showed the expected uridine auxotrophy and resistance to 5-FOA. Additionally, the genomic locus around the CRISPR target sites was sequenced to determine the exact circumstances around the gene disruption. To complete the genetic toolbox for , the gene of was tested as a suitable selection marker in one of the generated uridine auxotrophic strains.IMPORTANCEResearchers need an efficient tool for genomic manipulation to investigate the fundamental biology of mycoparasitism of and its correlation to secondary metabolites. We provide a protocol for transformation of and successfully demonstrated transformation of using a plasmid and genome editing applying a Cas9-RNP-based strategy. Simultaneously, we established two selection markers, the gene and gene from . By applying these methods, we give researchers the tools needed to investigate on a deeper level. Possible applications include activation of biosynthetic gene clusters of secondary metabolites to determine the biosynthetic pathway and biotechnological applications of these compounds.