CRISPR/Cas systems in fungal biotechnology: advancing high-value metabolite synthesis for industrial and food security applications.

Fungi serve as pivotal industrial platforms to produce essential compounds from pharmaceuticals like antibiotics and statins to diverse enzymes, organic acids and nutraceuticals. Fungi drive innovation across the medical, food and biofuel sectors through the synthesis of specialized pigments, flavors and bioactive molecules. Despite this potential, the development of efficient genetic toolkits has historically been slowed by low homologous recombination rates and biological complexity of diverse fungal taxa. The adaptation of CRISPR/Cas technologies including Cas9, Cas12a (Cpf1), and base editing has recently bypassed these bottlenecks and offers record precision in fungal genome engineering. This review provides a detailed analysis of current mechanistic approaches such as the use of ribonucleoprotein (RNP) complexes and specialized promoters for guide RNA expression. We have explored how these tools enable the multiplexed editing of genes and the fine-tuning of transcriptional modulation (i.e. CRISPR inactivation/activation) to redirect target metabolites for high industrial output by optimizing fungal physiology. Furthermore, the integration of CRISPR-based biosensors for the rapid, high-sensitivity detection of fungal pathogens, highlighting the dual role of these technologies in both production and food security has also been discussed.