CRISPR–Cas9 Applications in Fungi for Developing Energy and Biomaterials: Challenges and Perspectives

The increasing demand for both sustainable energy and bio-based materials has given rise to an imperative need for precision tools in the engineering of microbial platforms. Among these, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–Cas9, where Cas9 refers to CRISPR-associated protein 9, has revolutionized fungal biotechnology by enabling targeted genome editing with an unprecedented level of accuracy, efficiency, and scalability. Fungi, particularly filamentous species and oleaginous yeasts, are potential candidates that offer very promising perspectives for biofuel, organic acid, and functional biomaterial synthesis from renewable feedstocks. However, large and complex genomes, variable transformation efficiencies, and limited genetic toolkits have historically impeded their industrial development. This chapter provides a detailed overview of the transformative role of CRISPR–Cas9 in overcoming such limitations, explaining its applications in metabolic rewiring, pathway optimization, and regulatory circuit modulation across fungal systems such as Aspergillus nodulans, Trichoderma reesei, and Yarrowia lipolytica. Attention has been given to recent advances in multiplex editing, base editing, and CRISPRi/a strategies for increased lipid accumulation, cellulase production, and biopolymer biosynthesis. The chapter also addresses key challenges, including off-target effects, delivery systems, and intellectual property barriers, while proposing integrative frameworks that combine CRISPR with omics data, machine learning, and bioprocess engineering. By synthesizing current advances and future directions, this chapter positions CRISPR-enabled fungal platforms as pivotal agents in the development of next-generation bioenergy and biomaterials, aligned with circular economy and climate resilience goals.