CRISPR/Cas9 using a transient transformation system in Ceriporiopsis subvermispora

Ceriporiopsis subvermispora is a white-rot fungus with great potential for industrial and biotechnological applications, such as the pretreatment of lignocellulose in biorefineries, as it decomposes the lignin in the plant cell wall without causing severe cellulose degradation. A genetic transformation system was recently developed; however, gene-targeting experiments to disrupt or modify the gene(s) of interest remain challenging, and this is a bottleneck for further molecular genetic studies and breeding of C. subvermispora. Herein, we report efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene mutagenesis in this fungus. Two plasmids expressing Cas9 together with a different pyrG-targeting single-guide RNA were separately introduced into the monokaryotic C. subvermispora strain FP-90031-Sp/1, which frequently generated strains that exhibited resistance to 5-fluoroorotic acid and uridine/uracil auxotrophy. Southern blot analyses and genomic polymerase chain reaction followed by DNA sequencing of some mutants revealed that they were pyrG mutants. We also observed that hygromycin resistance of the pyrG mutants was frequently lost after repeated subcultivations, indicating that a maker-free genome editing occurred successfully. It is also suggested that a gene mutation(s) can be introduced via a transient expression of Cas9 and a single-guide RNA; this feature, together with high-frequency gene targeting using the CRISPR/Cas9 system, would be helpful for studies on lignocellulose-degrading systems in C. subvermispora. KEY POINTS: • Efficient plasmid-based CRISPR/Cas9 was established in C. subvermispora. • The mutations can be introduced via a transient expression of Cas9 and sgRNA. • A maker-free CRISPR/Cas9 is established in this fungus.