Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi

Iterative metabolic engineering of Fusarium fujikuroi has traditionally been hampered by its low homologous recombination efficiency and scarcity of genetic markers. Thus, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated proteins (Cas9) system has emerged as a promising tool for precise genome editing in this organism. Some integrated CRISPR/Cas9 strategies have been used to engineer F. fujikuroi to improve GA3 production capabilities, but low editing efficiency and possible genomic instability became the major obstacle. Herein, we developed a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in F. fujikuroi. This system, based on an autonomously replicating sequence, demonstrated the capability of a single plasmid harboring all editing components to achieve 100%, 75%, and 37.5% editing efficiency for single, double, and triple gene targets, respectively. Remarkably, even with a reduction in homologous arms to 50 bp, we achieved a 12.5% gene editing efficiency. By employing this system, we successfully achieved multicopy integration of the truncated 3‐hydroxy‐3‐methyl glutaryl coenzyme A reductase gene (tHMGR), leading to enhanced GA3 production. A key advantage of our plasmid‐based gene editing approach was the ability to recycle selective markers through a simplified protoplast preparation and recovery process, which eliminated the need for additional genetic markers. These findings demonstrated that the single‐plasmid CRISPR/Cas9 system enables rapid and precise multiple gene deletions/integrations, laying a solid foundation for future metabolic engineering efforts aimed at industrial GA3 production. Graphical and Lay Summary Iterative metabolic engineering of Fusarium fujikuroi has traditionally been hampered by its low homologous recombination efficiency and scarcity of genetic markers. In this study, the authors developed a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in F. fujikuroi, and demonstrated high editing efficiency and successful multiple gene deletions and integrations. This work is expected to significantly impact the production of secondary metabolites, particularly gibberellic acids, in industrial applications.