Gene targeting using pre-assembled Cas9 ribonucleoprotein and split-marker recombination in Pleurotus ostreatus

Until recently, classical breeding has been used to generate improved commercial mushroom strains; however, classical breeding remains to be laborious and time-consuming. In this study, we performed gene mutagenesis using Cas9 ribonucleoprotein (Cas9 RNP) as a plasmid-free genome editing in Pleurotus ostreatus, which is one of the most economically important cultivated mushrooms. The pre-assembled Cas9/sgRNA targeting pyrG was introduced into protoplasts of a wild-type monokaryotic P. ostreatus strain PC9, which resulted in a generation of strains exhibiting resistance to 5-fluoroorotic acid. Small insertions/deletions at the target site were identified using genomic PCR followed by sequencing. The results showed Cas9 RNP-assisted gene mutagenesis could be applied for the molecular breeding in P. ostreatus and in other edible mushroom strains. Furthermore, gene disruption via split-marker recombination using the Cas9 RNP system was also successfully demonstrated in wild-type P. ostreatus PC9. This method could overcome the disadvantages of NHEJ-deficiency in conventional studies with gene targeting, and also difficulty in gene targeting in various non-model agaricomycetes.