Protein kinase A gene knockout in Coprinopsis cinerea by CRISPR/Cas9 RNP complex system

Coprinopsis cinerea is a model mushroom for studying the developmental processes of homobasidiomycetous fungi. The development of C. cinerea depends on the sensing of environmental conditions, such as light, temperature, humidity and nutrients. The signal transduction pathway from the environmental condition sensors to the regulators and reproduction is an important research topic as the knowledge on it is still fragmentary. Protein kinases not only are crucial to the signal transduction, but also can be master switches of development. However, information on their role in basidiomycetous fungi is still limited. Using the effective gene-targeting of the current gene modification, a system of CRISPR/ Cas9 RNP with double strain template donor that contained a selective mark hygromycin cassette was developed in this study. In this system, CcPka1 gene knockout was successfully generated through homologues repair together with Cas9 RNP complexes. This was the first report to use CRISPR/ Cas9 RNP approach with C. cinerea. {Delta}CcPka1 mutants showed slow mycelial growth and rapid fruiting body induction compared to the wild type even when under high glucose condition. In addition, {Delta}CcPka1 mutants had significantly increased intracellular glucose level and decreased glycogen level. Moreover, {Delta}CcPka1 mutants regulated the gene expression of components in cAMP-PKA pathway. All these results demonstrated that PKA played an important role in the nutrient signal transduction and negatively regulated the fruiting body formation. This study also confirmed that GSK3 could be the downstream target of PKA, but their interaction requires further investigation.