Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli

Area of Science:

• Molecular Biology
• Mycology
• Biotechnology

Background:

• CRISPR-Cas9 technology offers powerful gene editing capabilities.
• Understanding DNA repair pathways is crucial for optimizing gene editing efficiency in fungi.

Purpose of the Study:

• To investigate the role of DNA repair pathways in CRISPR-Cas9 mediated gene editing in fungi.
• To develop novel tools and methods for efficient fungal gene editing.

Main Methods:

• Utilized CRISPR-Cas9 system with specific guide RNAs (sgRNAs) to induce DNA double-strand breaks (DSBs).
• Assessed the dependence of DSB repair on the non-homologous end-joining (NHEJ) pathway.
• Developed the TAPE tool to evaluate protospacer efficiency in Aspergillus nidulans.
• Employed single-stranded oligonucleotides (ssODNs) as repair templates in NHEJ-deficient strains.
• Designed a multiplexing vector system for delivering Cas9 and multiple sgRNAs.

Main Results:

• CRISPR-Cas9 mediated DSB repair in fungi is dependent on the NHEJ pathway.
• NHEJ-deficient strains enable highly efficient marker-free gene targeting.
• Single-stranded oligonucleotides serve as efficient repair templates for CRISPR-Cas9 induced DSBs in multiple Aspergillus species.
• Specific point mutations and gene deletions can be introduced with near 100% efficiency using ssODNs.
• Multiplexed gene editing, including multiple point mutations and gene insertions, achieved high efficiency in a single transformation.

Conclusions:

• The study provides a deeper understanding of CRISPR-Cas9 mediated gene editing mechanisms in filamentous fungi.
• Developed efficient and versatile tools for fungal gene editing, including marker-free gene targeting and multiplexing capabilities.
• The findings suggest broad applicability of ssODN-mediated repair for CRISPR-Cas9 gene editing across various filamentous fungi, paving the way for high-throughput applications.