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

Abstract

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, but also difficulty in gene targeting in various non-model agaricomycetes.

Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis

“…That is to say, the integration in genome is permanent, which does not cater to agriculture and food safety. On the contrary, an RNP complex formed by Cas9 and sgRNA, which are both transcribed and assembled in vitro; i.e., without restrictions on species, is an ideal strategy for genome editing due to its short half-life [ 32 , 43 , 44 ].…”

A Simple and Efficient CRISPR/Cas9 System Using a Ribonucleoprotein Method for Flammulina filiformis

“…The RNPs strategy involves the direct delivery of an in vitro Cas9/sgRNA complex. In basidiomycetes, there was a successful transformation of RNPs, such as in Schizophyllum commune [ 31 ] and P. ostreatus [ 32 ], but only a few mutants were obtained. In our study, the Cas9/sgRNA complex was used in F. filiformis for direct delivery into the protoplasts through PEG-mediated transformation.…”

Preassembled Cas9 Ribonucleoprotein-Mediated Gene Deletion Identifies the Carbon Catabolite Repressor and Its Target Genes in Coprinopsis cinerea

“…This approach is commonly used for genetic manipulations in filamentous fungi ( 34 ), but not in C. cinerea . Recently, genome editing using CRISPR/CRISPR-associated protein 9 (Cas9) has become popular in various fungi, including Agaricomycetes ( 35 – 42 ), and involves a report of using the split-marker together with the preassembled Cas9 ribonucleoproteins (Cas9 RNPs) in P. ostreatus ( 43 ).…”