Background

The basidiomycete fungus U. maydis is a model organism that has enabled important discoveries in topics like DNA repair and homologous recombination, mating, sexual development, secondary metabolism, filamentous growth, RNA transport and virulence (Holliday, 2004; Steinberg, 2007; Bakkeren et al., 2008; Holloman et al., 2008; Lanver et al., 2017; Niessing et al., 2018). U. maydis is a biotrophic plant pathogen that causes smut disease on maize. As a typical member of the large group of smut fungi its sexual development is intimately coupled with its ability to colonize plants. The popularity of U. maydis as a model organism resides in its short pathogenic life cycle that is completed in less than two weeks, its accessibility for forward and reverse genetics that includes the establishment of self-replicating plasmids (Tsukuda et al., 1988), the development of solopathogenic strains able to cause disease without mating and an available well-annotated genome (Kämper et al., 2006). In U. maydis, homologous recombination-based genome manipulations are very efficient, but become tedious when several genes have to be modified because the number of selectable markers is limited (Schuster et al., 2016). The most recent addition to the technical toolboxes for this organism has been the adaptation of the CRISPR-Cas9 genome editing technology (Schuster et al., 2016) and its development for multiplexed editing (Schuster et al., 2018). The CRISPR-Cas9 genome editing technology, developed from research in bacterial immune systems (Barrangou et al., 2007), has revolutionized molecular biology. The system allows efficient modification of almost any given sequence (Jinek et al., 2012) and has been established for a large number of organisms including filamentous fungi (Deng et al., 2017; Shi et al., 2017).

The CRISPR-Cas9 technology established for U. maydis is based on an all-in-one plasmid approach. A self-replicating plasmid (Tsukuda et al., 1988) is used as a backbone to provide the U. maydis codon-optimized Cas9 gene and the U6 promoter for sgRNA expression. This plasmid is unstable without selection and is rapidly lost (Figure 1 and Schuster et al. [2016]). The system proved very efficient for disruption of single genes reaching on average 70% of edited cells in progeny of one transformant (Schuster et al., 2016). A multiplexing version of the system was generated by using tRNA promoter-based expression cassettes that enabled the expression of several sgRNAs from the same plasmid (Schuster et al., 2018). This modification combined with elevated and longer expression of the Cas9 protein was used for the simultaneous disruption of five effector genes with 70% efficiency (Schuster et al., 2018). The multiplexed version of CRISPR-Cas9 has also been successfully applied for elucidating the contribution of oligopeptide transporters to virulence (Lanver et al., 2018).