Efficient genome editing using CRISPR/Cas9 technology and its application for identifying Sesquiterpene synthases involved in the biosynthesis of Steperoxides in Steccherinum ochraceum

CRISPR technology has been widely used for gene editing in various species，but the genetic manipulation in basidiomycete mushrooms is still notoriously difficult for unknown endogenous promoters and inefficient DNA delivery. Steccherinum ochraceum is a white rot basidiomycete fungus with abundant secondary metabolites and plays an important ecological role worldwide. To facilitate the study of gene function in S. ochraceum, an effective CRISPR/Cas9 system was successfully developed by identifying highly efficient endogenous promoters, and utilizing the Agrobacterium-transformation method. Two efficient endogenous RNA polymerase II promoters (Psogpd and Psotef1) and one efficient RNA polymerase III promoter (Pu6-d) were identified and characterized, with an editing efficiency of 61.5 % at the ura3 locus. Using this optimized system, the sesquiterpene gene A0064, which could produce 10 possible sesquiterpenes in the heterologous expression system of A. oryzae, was knocked out to obtain A0064 knockout strain S. ochraceum (∆A0064). Steperoxide A could not be detected in S. ochraceum (∆A0064), demonstrating that A0064 was the only enzyme responsible for the biosynthesis of β-chamigrene (the sesquiterpene skeleton of steperoxide A) in S. ochraceum. This efficient system will enable precise targeting and multiplex editing of S. ochraceum genes, facilitating functional studies of genes involved in lignin degradation and natural product biosynthesis in S. ochraceum, and providing some valuable guidance for gene editing in tens of thousands of macrofungi.