Gene targeting of dikaryotic Pleurotus ostreatus nuclei using the CRISPR/Cas9 system

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene targeting is a promising method used in molecular breeding. We recently reported the successful introduction of this method in the monokaryotic Pleurotus ostreatus (oystermushroom), PC9. However, considering their application in mushroom breeding, dikaryotic strains (with targeted genemutations in both nuclei) need to be generated. This is laborious and time-consuming because a classical crossing technique is used. Herein, we report a technique that targets both nuclei of dikaryotic P. ostreatus, PC9x#64 in a transformation experiment using plasmid-based CRISPR/Cas9, with the aim of developing a method for efficient and rapid molecular breeding. As an example, we targeted strains with low basidiospore production ability through the meiosis-related genes mer3 or msh4. Four different plasmids containing expression cassettes for Cas9 and two different gRNAs targeting mer3 or msh4 were constructed and separately introduced into PC9x#64. Eight of the 38 dikaryotic transformants analyzed produced no basidiospores. Genomic PCR suggested that msh4 or mer3 mutations were introduced into both nuclei of seven out of eight strains. Thus, in this study, we demonstrated simultaneous gene targeting using our CRISPR/Cas9 system, which may be useful for the molecular breeding of cultivated agaricomycetes.

Automated summary

In this study, we reported a method for simultaneously targeting both nuclei of dikaryotic P. ostreatus PC9x#64 using plasmid-based CRISPR/Cas9, aiming to develop an efficient and rapid molecular breeding method. By targeting the meiosis-related genes mer3 and msh4, mutations were successfully introduced and expressed in both nuclei.