Pre-assembled Cas9 ribonucleoprotein-mediated gene deletion identifies the carbon catabolite repressor and its target genes in Coprinopsis cinerea

Abstract

Cre1 is an important transcription factor that regulates carbon catabolite repression (CCR) and is widely conserved across fungi. This gene has been extensively studied in several Ascomycota species, whereas its role in gene expression regulation in the Basidiomycota remains poorly understood. Here, we identified and investigated the role of cre1 in Coprinopsis cinerea, a basidiomycete model mushroom that can efficiently degrade lignocellulosic plant wastes. We used a rapid and efficient gene deletion approach based on PCR-amplified split-marker DNA cassettes together with in-vitro assembled Cas9-guide RNA ribonucleoproteins (Cas9-RNPs) to generate C. cinerea cre1 gene deletion strains. Gene expression profiling of two independent C. cinerea cre1 mutants showed significant deregulation of carbohydrate metabolism, plant cell wall degrading enzymes (PCWDEs), plasma membrane transporter-related and several transcription factor encoding genes, among others. Our results support the notion that, similarly to reports in the ascomycetes, Cre1 of C. cinerea orchestrates CCR through a combined regulation of diverse genes, including PCWDEs, transcription factors that positively regulate PCWDEs and membrane transporters which could import simple sugars that can induce the expression of PWCDEs. Somewhat paradoxically, though in accordance with other Agaricomycetes, genes related to lignin degradation were mostly downregulated in cre1 mutants, indicating they fall under different regulation than other PCWDEs. The gene deletion approach and the data presented in this paper expand our knowledge of CCR in the Basidiomycota and provide functional hypotheses on genes related to plant biomass degradation. ImportanceMushroom-forming fungi include some of the most efficient degraders of lignocellulosic plant biomass. They degrade dead plant materials by a battery of lignin-, cellulose-, hemicellulose- and pectin-degrading enzymes, the encoding genes of which are under tight transcriptional control. One of the highest-level regulation of these metabolic enzymes is known as carbon catabolite repression, which is orchestrates by the transcription factor Cre1, and ensures that costly lignocellulose-degrading enzyme genes are expressed only when simple carbon sources (e.g. glucose) are not available. Here, we identified the Cre1 ortholog in a litter-decomposer Agaricomycete, Coprinopsis cinerea, knocked it out and characterized transcriptional changes in the mutants. We identified several dozen lignocellulolytic enzyme genes as well as membrane transporters and other transcription factors as putative target genes. These results extend knowledge on carbon catabolite repression to litter decomposer Basidiomycota.