Clustered Regularly Interspaced Palindromic Repeats–Cas Genome-Editing Systems in Filamentous Fungi: Current Tools, Optimization Strategies, and Future Perspectives

Abstract Filamentous fungi are important microbial cell factories for producing antibiotics, organic acids, enzymes, and other valuable products, with broad applications in food, agriculture, and medicine. However, inefficient and imprecise genome editing remains a major bottleneck limiting rational strain engineering. This review summarizes major genome-editing tools in filamentous fungi, including Cre-loxP, TALENs, and CRISPR–Cas systems, with emphasis on their mechanisms, advantages, and limitations. We highlight CRISPR–Cas9 as the predominant platform and discuss optimization strategies involving sgRNA design, engineered Cas9 variants, promoter engineering, AMA1-based replicative plasmids, and ribonucleoprotein delivery. Emerging technologies, including base editing, prime editing, Cas12a, and Mad7, are also discussed for their potential to broaden editing capabilities. Finally, we outline future perspectives focusing on extending the application of existing genome-editing systems in filamentous fungi and discovering novel genome-editing tools from large-scale omics data sets. This review will accelerate the development of filamentous fungi as versatile platforms for biomanufacturing.