Efficient gene editing in Neurospora crassa with CRISPR technology

Area of Science:

• Molecular Biology
• Mycology
• Biotechnology

Background:

• Efficient gene editing is crucial for understanding cellular processes and organism engineering.
• RNA-guided CRISPR/Cas9 technology offers powerful gene editing capabilities.
• CRISPR/Cas9 had not been previously tested in the model filamentous fungus Neurospora crassa.

Purpose of the Study:

• To demonstrate the efficacy of the CRISPR/Cas9 system for gene editing in Neurospora crassa.
• To establish methods for gene replacement and reporter gene insertion in Neurospora crassa.
• To facilitate genetic perturbations in filamentous fungi.

Main Methods:

• Utilized Cas9 endonuclease and chimeric guide RNA (gRNA) for gene editing.
• Replaced the endogenous promoter of the CLR-2 gene with the beta-tubulin promoter.
• Inserted a codon-optimized firefly luciferase reporter gene under the GSY-1 promoter at the CSR-1 locus.

Main Results:

• Successfully achieved efficient gene replacement in Neurospora crassa using the CRISPR/Cas9 system.
• The beta-tubulin promoter-driven CLR-2 strain exhibited increased cellulase expression.
• The GSY-1 promoter-driven luciferase reporter strain allowed for easy screening via bioluminescence assays.

Conclusions:

• The CRISPR/Cas9 system is highly effective in Neurospora crassa.
• This technology can be adapted for use in Neurospora natural isolates and other filamentous fungi.
• CRISPR/Cas9 tools will significantly benefit the filamentous fungal research community for genetic studies.