The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments

Abstract

Gene editing is a promising alternative to traditional breeding for the generation of new mushroom strains. However, the current approach frequently uses Cas9-plasmid DNA to facilitate mushroom gene editing, which can leave residual foreign DNA in the chromosomal DNA raising concerns regarding genetically modified organisms. In this study, we successfully edited pyrG of Ganoderma lucidum using a preassembled Cas9-gRNA ribonucleoprotein complex, which primarily induced a double-strand break (DSB) at the fourth position prior to the protospacer adjacent motif. Of the 66 edited transformants, 42 had deletions ranging from a single base to large deletions of up to 796 bp, with 30 being a single base deletion. Interestingly, the remaining 24 contained inserted sequences with variable sizes at the DSB site that originated from the fragmented host mitochondrial DNA, E. coli chromosomal DNA, and the Cas9 expression vector DNA. The latter two were thought to be contaminated DNAs that were not removed during the purification process of the Cas9 protein. Despite this unexpected finding, the study demonstrated that editing G. lucidum genes using the Cas9-gRNA complex is achievable with comparable efficiency to the plasmid-mediated editing system.

Pleurotus ostreatus as a model mushroom in genetics, cell biology, and material sciences

“…The RNP-based method has been reported for some agaricomycetes, such as S. commune (Jan Vonk et al 2019 ), P. ostreatus (Boontawon et al 2021b ), Dichomitus squalens (Kowalczyk et al 2021 ), F. filiformis (Liu et al 2022b ), C. cinerea (Pareek et al 2022 ), and G. lucidum (Eom et al 2023 ) (Table 1 ). Technically, this protocol can be applied to species lacking a suitable selection marker gene, provided that protoplasts can be generated efficiently.…”

“…However, at present, the efficiency of genome editing using RNP-based methods may be lower than that of DNA-based methods (Boontawon et al 2021b ). The major challenge of the RNP-based method is the lack of an efficient selection system; this method has been applied only to a special target gene whose disruption makes it possible to screen for, such as pyrG (Boontawon et al 2021b ; Kowalczyk et al 2021 ; Eom et al 2023 ) or fcy1 (Boontawon et al 2023 ), unless a foreign selection marker was also introduced (Jan Vonk et al 2019 ; Boontawon et al 2021b ; Kowalczyk et al 2021 ; Pareek et al 2022 ). To overcome this problem, RNP-dependent CRISPR/Cas9 with two target genes, one for screening and the other the gene of interest, was developed in P. ostreatus (Boontawon et al 2023 ).…”

“…RNP-based methods can introduce mutations at the target site in a foreign DNA-independent manner, which may be beneficial for isolating strains free of heterogeneous DNA sequences. However, in G. lucidum , frequent integration of contaminated and mitochondrial DNA fragments at the target site has been observed in RNP-dependent genome editing (Eom et al 2023 ). Integration of unexpected DNA sequences at the target and off-target sites could be a fatal problem in CRISPR/Cas9, both in DNA- and RNP-dependent protocols, although successful isolation of small insertion/deletion mutations has been reported in P. ostreatus (Boontawon et al 2023 ).…”

An Efficient CRISPR/Cas9 Genome Editing System for a Ganoderma lucidum Cultivated Strain by Ribonucleoprotein Method

“…The G. lucidum dikaryotic strain GL3315 is from the Sorak Mountains located in the northwestern part of Korea. Eom et al successfully constructed a CRISPR/Cas9 genome editing system for the monokaryotic strain of GL3315 using ribonucleoproteins (RNPs) assembled by the Cas9 protein and sgRNA [13], with the editing efficiencies of 0, 0.33, and 22 mutants/10 7 protoplasts for different target sites, and its transformation system was supplemented with 0.90% of Triton X-100.…”

“…The CRISPR/Cas9 systems of G. lucidum are constructed using the orotidine 5monophosphate decarboxylase gene (ura3) as the target gene [5,[10][11][12][13]. The ura3 gene of G. lucidum is involved in catalyzing a key reaction in the synthesis of uracil and can convert 5-fluoroorotic acid (5-FOA) into the toxic compound 5-fluorouracil (a suicide inhibitor), causing cell death [14].…”