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

Abstract

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 (oyster mushroom), PC9. However, considering their application in mushroom breeding, dikaryotic strains (with targeted gene mutations 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, PC9×#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 PC9×#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.

Experimental evidence that lignin‐modifying enzymes are essential for degrading plant cell wall lignin byPleurotus ostreatususingCRISPR/Cas9

“…Next, we examined whether a mutation was introduced into each of the four genes (vp2, vp3, mnp3, or mnp6) in each transformant using genomic PCR experiments. Our previous studies showed that the introduced plasmid(s) were often integrated at the target site of sgRNA in some of the P. ostreatus mutants obtained from the pCcPef3-126-based CRISPR/Cas9 (Boontawon, Nakazawa, Horii, et al, 2021;Boontawon, Nakazawa, Inoue, et al, 2021;Koshi et al, 2022;Yamasaki et al, 2022). This usually caused insufficient amplification from the mutants with large insertions due to the requirement of a longer extension time than the fragment amplified from the parental control strain.…”

Application progress of CRISPR/Cas9 genome-editing technology in edible fungi

“…However, these classical breeding methods are laborious and time-consuming. Thus, methodologies for more time- and cost-effective molecular breeding are needed (Yamasaki et al, 2022 ). The implementation of the CRISPR/Cas9 system in P. ostreatus will undoubtedly facilitate its molecular breeding.…”

“…In addition to the monokaryotic P. ostreatus strain PC9, CRISPR/Cas9 has also been successfully performed on the dikaryotic P. ostreatus . In 2022, Yamasaki et al ( 2022 ) reported a plasmid-based CRISPR/Cas9 technique for transforming both nuclei of dikaryotic P. ostreatus , PC9 × #64, to generate strains with low basidiospore production ability. Meiosis-related genes mer3 or msh4 were selected as target genes to design two different sgRNAs.…”

“…The mycelia of F. velutipes monokaryon "Dan3" transformed by the Agrobacterium-mediated method were used to obtain transformants. However, the transformation rate was low, only 6.84% (Liu et al, 2017); 5 years later, Liu J. et al (2022) first established a CRISPR/Cas9 genome-editing system in the mushroom F. velutipes using a fast, accurate, and transgenefree RNP complex delivery technology (Wang P. A. et al, 2022), greatly improving the targeting efficiency to 100%. In this study, a CRISPR/Cas9 genome-editing method based on in vitro assembled Cas9 and RNPs was developed, which avoids the use of any exogenous DNA, thus saving time and manpower in plasmid construction.…”

Structural Analysis of the A Mating Type Locus and Development of the Mating Type Marker of Agaricus bisporus var. bisporus

“…Molecular breeding by modifying genomic sequences to alter the function and expression of genes of interest [ 1 ] or by introducing foreign DNA to the host chromosomal sequence [ 2 , 3 , 4 ] has been attempted with some mushrooms; however, this approach is not applicable to edible mushrooms because the mushrooms generated in this way are essentially genetically modified organisms (GMOs). Nonetheless, a new technology in molecular breeding has emerged recently, namely CRISPR-Cas9 gene-editing technology, which enables targeted modification of the genes or DNA sequences of certain functions [ 5 , 6 , 7 , 8 ]. It has been applied to generate a new strain of A. bisporus that is resistant to browning via gene-editing of polyphenol oxidase (PPO) [ 9 ].…”