Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 57 In order to overcome these limitations, a research team specifically selected the widely cultivated monokaryotic strain L1 of "Shanghai Agricultural No. 1" G. lucidum, established and optimized a CRISPR/Cas9 system based on RNP (i.e., ribonucleoprotein) (Tan et al., 2023). In this system, the researchers used ura3 as the target gene and achieved a considerable editing efficiency: more than 35 mutants were successfully obtained for every 107 protoplasts. This shows that the system is stable in inducing DNA double-strand breaks and can complete gene editing through repair mechanisms such as NHEJ and MMEJ. In addition, the study further edited key genes related to triterpenoid metabolism, such as cyp512a3 and cyp5359n1, providing new tools for regulating triterpenoid synthesis. In another experiment, the researchers tried to add introns to the promoter region of the Cas9 gene, which improved the overall editing efficiency of the system and ultimately achieved a 36.7% ura3 gene deletion rate in G. lucidum(Figure 2) (Liu et al., 2020). These studies provide a technical basis for subsequent functional gene verification and directed breeding, and also make the genome manipulation of G. lucidummore feasible. Figure 2 An effective platform for disruption and deletion of target genes inG. lucidum(Adopted from Liu et al., 2020) Image caption: The figure illustrates the overall process and results of gene knockout and fragment deletion in G. lucidumusing the CRISPR/Cas9 system. It describes the design and steps of two different Cas9 vectors (opCas9 and intron-opCas9) for gene editing, including protoplast transformation, sgRNA targeting design, gene disruption, and fragment deletion processes. The results indicate the superiority of the Cas9 system with an intron, significantly enhancing the efficiency of gene disruption and fragment deletion, validating the effectiveness of the CRISPR/Cas9 system for precise gene editing inG. lucidum(Adopted from Liu et al., 2020) 6.2 Overexpression of key enzymes in triterpenoid pathways In order to increase the content of triterpenoids in G. lucidum, researchers have tried to overexpress key enzymes in the triterpene biosynthesis pathway. In a study, scientists identified for the first time the transcription factor GlbHLH5 as an important regulator of triterpene synthesis and regarded it as a potential metabolic engineering target (Xu et al., 2022a). The experiment found that methyl jasmonic acid (MeJA) treatment not only significantly promoted the accumulation of triterpenoids, but also upregulated the expression level of GlbHLH5, thereby further enhancing its regulatory effect. By constructing transgenic G. lucidum strains that overexpressed and silenced GlbHLH5, the researchers found that the triterpene production of the former increased significantly, while the latter showed a phenomenon of blocked synthesis (Figure 3), verifying its key role.
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