CRISPR/Cas9 using a transient transformation system in Ceriporiopsis subvermispora

Area of Science:

• Mycology
• Molecular Biology
• Biotechnology

Background:

• Ceriporiopsis subvermispora is a white-rot fungus valuable for lignocellulose pretreatment in biorefineries due to selective lignin degradation.
• Existing genetic tools for C. subvermispora are limited, hindering molecular studies and strain improvement.
• Efficient gene targeting is essential for understanding and enhancing the fungus's biotechnological capabilities.

Purpose of the Study:

• To establish an efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system for gene mutagenesis in Ceriporiopsis subvermispora.
• To demonstrate the feasibility of maker-free genome editing in this fungus.
• To facilitate future molecular genetic studies and breeding of C. subvermispora.

Main Methods:

• Development of CRISPR/Cas9 plasmids with pyrG-targeting single-guide RNA for transformation into C. subvermispora.
• Selection of transformants based on 5-fluoroorotic acid resistance and uridine/uracil auxotrophy.
• Confirmation of gene mutations using Southern blot analysis, genomic PCR, and DNA sequencing.

Main Results:

• Successfully generated pyrG mutants in C. subvermispora via CRISPR/Cas9-mediated gene editing.
• Demonstrated transient expression of Cas9 and single-guide RNA leads to successful gene mutation.
• Observed frequent loss of hygromycin resistance, indicating successful maker-free genome editing.

Conclusions:

• An efficient plasmid-based CRISPR/Cas9 system is established for C. subvermispora.
• The CRISPR/Cas9 system allows for maker-free gene editing through transient expression.
• This technology will accelerate research on lignocellulose degradation and fungal biotechnology.