Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger

Area of Science:

• Molecular Biology
• Genetics
• Mycology

Background:

• CRISPR/Cas9 is a key tool for precise genome editing in filamentous fungi.
• Efficient guide RNA (gRNA) delivery is a major challenge for CRISPR mutagenesis in Aspergillus species.

Purpose of the Study:

• To develop and validate a novel method for gRNA transcription using endogenous tRNA promoters for CRISPR/Cas9 genome editing in Aspergillus niger.
• To enhance the efficiency of gene mutation and replacement in Aspergillus species.

Main Methods:

• Co-transformation of a cas9-expressing plasmid with linear DNA encoding gRNA driven by tRNA promoters.
• Expression of both cas9 and gRNA from a single extra-chromosomal plasmid.
• Utilizing homologous recombination with DNA templates for gene replacement, comparing kusA+ and kusA- strains.

Main Results:

• 36 out of 37 tested tRNA promoters successfully generated intended mutations in A. niger.
• Gene mutation efficiency reached 97% when cas9 and gRNA were on a single plasmid.
• Gene replacement efficiency was ~42% in kusA+ strains and >90% in kusA- strains.

Conclusions:

• tRNA promoter-mediated gRNA expression provides a reliable and highly efficient system for CRISPR/Cas9 genome editing in Aspergillus niger.
• This method overcomes previous limitations in gRNA delivery for Aspergillus mutagenesis.
• The developed system significantly improves gene editing outcomes in filamentous fungi.