Werner, J.; Zuo, W.; Doehlemann, G.

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has become the state of art for mutagenesis in filamentous fungi. Here, we describe a RNP-mediated CRISPR/Cas9 for mutagenesis in Sporisorium reilianum. The efficiency of the method was tested in vitro with a cleavage assay as well as in vivo with a GFP expressing S. reilianum strain. We applied this method to generate frameshift-, knock-in- and knock-out mutants in S. reilianum without a resistance marker by using an auto-replicating plasmid for selection. The RNP-mediated CRISPR/Cas9 increased the mutagenesis efficiency and firstly enables a marker-free genome editing in S. reilianum.