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Updated: Mar 28, 2026
CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis
Published on: June 9, 2020
Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus
Chi Zhang1, Xiuhua Meng1, Xiaolei Wei1
1Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
Abstract:
Filamentous fungi have a dominant nonhomologous-end joining (NHEJ) DNA repair pathway, which results in the majority of transformed progenies having random heterologous insertion mutagenesis. Thus, lack of a versatile genome-editing tool prevents us from carrying out precise genome editing to explore the mechanism of pathogenesis. Moreover, clinical isolates that have a wild-type ku80 background without any selection nutrition marker especially suffer from low homologous integration efficiency. In this study, we have established a highly efficient CRISPR mutagenesis system to carry out precise and efficient in-frame integration with or without marker insertion with approximately 95-100% accuracy via very short (approximately 35-bp) homology arms in a process referred to as microhomology-mediated end joining (MMEJ). Based on this system, we have successfully achieved an efficient and precise integration of an exogenous GFP tag at the predicted site without marker insertion and edited a conidial melanin gene pksP and a catalytic subunit of calcineurin gene cnaA at multiple predicted sites with or without selection marker insertion. Moreover, we found that MMEJ-mediated CRISPR-Cas9 mutagenesis is independent of the ku80 pathway, indicating that this system can function as a powerful and versatile genome-editing tool in clinical Aspergillus isolates.
Insights
This study introduces a CRISPR-Cas9 system utilizing microhomology-mediated end joining (MMEJ) for precise genome editing in filamentous fungi. This efficient method overcomes limitations of random insertions, enabling accurate gene modification in clinical isolates.
Area of Science:
- Molecular Biology
- Mycology
- Genetics
Background:
- Filamentous fungi predominantly use nonhomologous-end joining (NHEJ) for DNA repair, leading to random gene insertions.
- The lack of precise genome-editing tools hinders the study of fungal pathogenesis mechanisms.
- Clinical fungal isolates with wild-type ku80 and no selection markers exhibit low homologous integration efficiency.
Purpose of the Study:
- To establish a highly efficient CRISPR mutagenesis system for precise genome editing in filamentous fungi.
- To enable markerless in-frame gene integration with high accuracy.
- To develop a versatile genome-editing tool for clinical fungal isolates.
Main Methods:
- Developed a CRISPR-Cas9 system leveraging microhomology-mediated end joining (MMEJ).
- Utilized very short homology arms (approximately 35 bp) for precise integration.
- Applied the system for GFP tagging and editing of pksP and cnaA genes, with and without selection markers.
Main Results:
- Achieved 95-100% accuracy for precise in-frame integration using MMEJ.
- Successfully integrated an exogenous GFP tag without a marker at the target site.
- Edited multiple genes (pksP, cnaA) with high precision, with or without selection markers.
- Demonstrated MMEJ-mediated mutagenesis is independent of the ku80 pathway.
Conclusions:
- The MMEJ-mediated CRISPR-Cas9 system provides a powerful and versatile tool for precise genome editing in filamentous fungi.
- This system overcomes the limitations of NHEJ and is effective in clinical Aspergillus isolates, regardless of ku80 background.
- Enables precise gene modification, facilitating research into fungal pathogenesis and genetic manipulation.
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