CRISPR/Cas9 Ribonucleoprotein-Mediated Mutagenesis in .

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has become the state of the art for mutagenesis in filamentous fungi. Here, we describe a ribonucleoprotein complex (RNP)-mediated CRISPR/Cas9 for mutagenesis in . The efficiency of the method was tested in vitro with a cleavage assay as well as in vivo with a GFP-expressing strain. We applied this method to generate frameshift- and knock-out mutants in without a resistance marker by using an auto-replicating plasmid for selection. The RNP-mediated CRISPR/Cas9 increased the mutagenesis efficiency, can be applied for all kinds of mutations, and enables a marker-free genome editing in . Key features • First CRISPR/Cas9 application in • Generation of mutants without genomic integration of resistance marker. • Allows the generation of multiple gene knockouts as well as deletion of large genomic regions.