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Dual sgRNA-directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

Summary

This research developed an improved genetic engineering tool for modifying medicinal mushrooms, specifically Ganoderma lucidum. The scientists created a more efficient way to edit genes in these fungi using CRISPR technology, which could lead to better understanding and potential enhancement of their medicinal properties. Impacts on everyday life: – Could lead to improved production of medicinal mushroom compounds – May help develop more effective natural medicines – Could enable creation of enhanced mushroom strains with better therapeutic properties – May contribute to more sustainable and efficient mushroom cultivation methods

Background

Ganoderma lucidum is an important medicinal mushroom used in traditional Chinese medicine for millennia to improve health and longevity. While its genome has been sequenced, functional characterization of genes remains challenging due to lack of adequate molecular genetic tools. Current genetic manipulation methods like gene silencing only achieve partial suppression, making targeted gene deletion capabilities highly desirable.

Objective

To develop an improved CRISPR/Cas9 system for efficient gene disruption and deletion in G. lucidum by adding an intron upstream of the Cas9 gene, and to establish a dual sgRNA-directed approach for targeted gene deletion.

Results

The addition of an intron was necessary for efficient expression of heterologous genes in G. lucidum. The improved CRISPR/Cas9 system with intron achieved a ura3 disruption frequency of 14-18 in 10^7 protoplasts, which was 10.6 times higher than without the intron. Using dual sgRNAs, they achieved a 36.7% deletion frequency for ura3 and successfully deleted the GL17624 gene with 13.3% efficiency.

Conclusion

The researchers successfully developed an improved CRISPR/Cas9 system for G. lucidum by incorporating an intron, significantly increasing gene disruption efficiency. They also established the first dual sgRNA-directed gene deletion method in non-model mushrooms. This technology provides a powerful platform for generating gene deletion mutants and will facilitate functional genomic studies in G. lucidum.
  • Published in:Microbial Biotechnology,
  • Study Type:Laboratory Research,
  • Source: 10.1111/1751-7915.13534
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