Skip to main content
ISMPMI Congress 2025 Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.

Poster

Session: Session 2 Presenting Authors at Posters (Odds)

P-191 - A Streamlined Protocol for Small-scale Protoplast Generation and CRISPR/Cpf1-mediated Genome Editing in Fusarium oxysporum

Wednesday, July 16, 2025
13:30 - 15:15  
Location: Confex Hall 3
Engineering plant-microbe traits

    Author(s)

  • JZ

    Jun-Ze Zheng

    Program in Plant Health Care, Academy of Circular Economy, National Chung Hsing University
    Nantou, Nantou, Taiwan (Republic of China)

  • SH

    Szu-Chieh Huang

    Program in Plant Health Care, Academy of Circular Economy, National Chung Hsing University
    Nantou, Nantou, Taiwan (Republic of China)

    • Presenting Author(s)

  • TC

    Tao-Ho Chang, PhD

    Assistant Professor
    Program in Plant Health Care, Academy of Circular Economy, National Chung Hsing University
    Nantou City, Nantou, Taiwan (Republic of China)

Efficient genetic manipulation of the vital plant pathogen Fusarium oxysporum remains challenging despite advances in gene editing technologies. This study presents an optimised small-scale protoplast preparation protocol coupled with a CRISPR/Cpf1 ribonucleoprotein (RNP) delivery system for precise genome editing. The streamlined method reduces culture volume and enzyme requirements while maintaining high-quality protoplasts. Four osmotic stabilisers were tested, with a solution containing Na₂HPO₄, CaCl₂, and NaCl at optimised pH identified as most effective for protoplast generation and viability. Using the URA5 gene as a target, successful CRISPR/Cpf1 editing was demonstrated with 5-FOA selection. Homology-directed repair efficiency was analysed using EGFP expression cassettes with varying homology arm lengths (20 bp, 100 bp, and 1000 bp). Longer homology arms showed substantially higher editing efficiency with precise gene replacement, while shorter arms demonstrated proportionally reduced efficiency. Interestingly, shorter homology arms showed evidence of random insertion. This protocol enhances the feasibility of functional genomics studies in F. oxysporum and provides insights for designing gene editing strategies in filamentous fungi. Ongoing work aims to further refine this methodology and expand its application for developing non-pathogenic strains with potential uses in sustainable agriculture.