The CRISPR/Cas9 system has become a popular approach to genome editing. Compared with the plasmid-dependent CRISPR system, the ribonucleoprotein (RNP) complex formed by the in vitro assembly of Cas9 and single-guide RNA (sgRNA) has many advantages. However, only a few examples have been reported and the editing efficiency has been relatively low. In this study, we developed and optimized an RNP-mediated CRISPR/Cas9 genome editing system for the monokaryotic strain L1 from the Ganoderma lucidum cultivar ‘Hunong No. 1’. On selective media containing 5-fluoroorotic acid (5-FOA), the targeting efficiency of the genomic editing reached 100%. The editing efficiency of the orotidine 5′-monophosphate decarboxylase gene (ura3) was greater than 35 mutants/10⁷ protoplasts, surpassing the previously reported G. lucidum CRISPR systems. Through insertion or substitution, 35 mutants introduced new sequences of 10–569 bp near the cleavage site of ura3 in the L1 genome, and the introduced sequences of 22 mutants (62.9%) were derived from the L1 genome itself. Among the 90 mutants, 85 mutants (94.4%) repaired DNA double-strand breaks (DSBs) through non-homologous end joining (NHEJ), and five mutants (5.6%) through microhomology-mediated end joining (MMEJ). This study revealed the repair characteristics of DSBs induced by RNA-programmed nuclease Cas9. Moreover, the G. lucidum genes cyp512a3 and cyp5359n1 have been edited using this system. This study is of significant importance for the targeted breeding and synthetic metabolic regulation of G. lucidum.

Ganoderic acids (GAs) are major functional components of Ganoderma lucidum. The study aimed to breed a new G. lucidum strain with increased contents of individual GAs. Two mating-compatible monokaryotic strains, G. 260125 and G. 260124, were successfully isolated from the dikaryotic G. lucidum CGMCC 5.0026 via protoplast formation and regeneration. The Vitreoscilla hemoglobin gene (vgb) and squalene synthase gene (sqs) were overexpressed in the monokaryotic G. 260124 and G. 260125 strain, respectively. Mating between the G. 260124 strain overexpressing vgb and the G. 260125 strain overexpressing sqs resulted in the formation of the new hybrid dikaryotic G. lucidum strain sqs-vgb. The maximum contents of ganoderic acid (GA)-T, GA-Me, and GA-P in the fruiting body of the mated sqs-vgb strain were 23.1, 15.3, and 39.8 μg/g dry weight (DW), respectively, 2.23-, 1.75-, and 2.69-fold greater than those in G. lucidum 5.0026. The squalene and lanosterol contents increased 2.35- and 1.75-fold, respectively, in the fruiting body of the mated sqs-vgb strain compared with those in the G. lucidum 5.0026. In addition, the maximum expression levels of the sqs and lanosterol synthase gene (ls) were increased 3.23- and 2.13-fold, respectively, in the mated sqs-vgb strain. In summary, we developed a new G. lucidum strain with higher contents of individual GAs in the fruiting body by integrating genetic engineering and mono–mono crossing.