Development of a CRISPR/Cas9 Genome Editing System in Dikaryotic Ganoderma lucidum for Targeting Key CYP450 Gene Involved in Triterpenoid Synthesis

Currently, most research on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing in edible fungi focuses on monokaryotic strains. However, the biological mechanisms in a monokaryotic state often do not accurately reflect the actual physiological and metabolic conditions of dikaryotic strains. Therefore, this study used two mating-type-compatible monokaryotic strains, L1 and L2, isolated from Ganoderma lucidum 'Hunong No.1' G0119, and employed an RNP (ribonucleoprotein)-based CRISPR/Cas9 system to successfully knock out the cyp512a3 gene in strain L2, resulting in the edited strain L2-KO-cyp512a3. The strain was single-crossed with the previously edited L1 strain L1-KO-cyp512a3 in our laboratory to obtain a dikaryotic editing strain that was homozygous at the cyp512a3 locus, named G0119-KO-cyp512a3. UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) analysis showed that compared to the starting strain G0119, the dikaryotic editing strain exhibited varying degrees of reduction in the content of eight types of ganoderic acids, including ganoderic acid Me, ganoderic acid P, ganoderic acid T1, etc., with the reduction ranging from 30.5% to 80.1%. To further validate the function of cyp512a3, we overexpressed this gene in the L1 strain. The results showed that the contents of ganoderic acid Mk, ganoderic acid S, ganoderic acid T, and ganoderic acid R in the mycelium were 0.548 ± 0.020, 1.780 ± 0.028, 2.416 ± 0.148, and 0.281 ± 0.016 mg/g (dry weight), which were 1.5 times, 1.3 times, 1.3 times, and 1.3 times that of G0119, respectively. By integrating the results of gene knockout and overexpression, it can be clearly established that cyp512a3 is a key cytochrome P450 gene regulating the biosynthesis of ganoderic triterpenoids in Ganoderma lucidum. This study not only establishes, for the first time, a homologous recombination-based gene editing system in dikaryotic strains of <i>Ganoderma lucidum</i>, but also provides a research paradigm based on a dikaryon-editing tool for investigating key life traits of other edible fungi.