Targeted gene knock-in reduces variation between transformants in the mushroom-forming fungus Schizophyllum commune

Abstract

Gene integration in mushroom-forming fungi currently occurs by the ectopic integration of a plasmid. The locus of integration is unpredictable and, problematically, this generally results in a high variability in gene expression and phenotypes between the transformants. Here, we developed an approach for targeted gene integration (knock-in) in the basidiomycete Schizophyllum commune by replacing a 75-bp non-coding region of the genome with a selection marker and an arbitrary gene of interest using CRISPR-Cas9 ribonucleoproteins. To assess the suitability of our method, we compared targeted integration and ectopic integration of the gene encoding the red fluorescent protein dTomato. Targeted integration resulted in a higher average fluorescence intensity and less variability between the transformants. This method may be applied to any gene construct and may therefore greatly increase the efficiency of functional gene analysis in S. commune.