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.

Snowball: a novel gene family required for developmental patterning in fruiting bodies of mushroom-forming fungi (Agaricomycetes)

“…To characterize snb1 using reverse genetics, we applied an RNP-complex-based CRISPR/Cas9 system (34), and obtained three independent mutants, displaying the same phenotype. While we could not observe any significant changes in the growth of vegetative mycelia, the development of the fruiting bodies showed severe deficiencies, reminiscent of snowballs, which were unable to form a fully structured cap, stipe or gills, resulting in a lack of sporulation.…”