EDGE-FGT: Enabling CRISPR-Based Functional Genomics of Non-Model, Difficult-To-Manipulate Fungi

Mapping genes to functions at scale is fundamental to advancing biology and biotechnology, enabling innovations in sustainable biomanufacturing, environmental bioremediation, and human health. Fungi play essential roles in producing chemicals, materials, enzymes, food ingredients, and other high-value products, yet they also include pathogens that cause life-threatening infections and are increasingly resistant to antifungal drugs. Although genome sequencing has uncovered vast genetic diversity across fungal species, the functions of many genes remain unknown because most non-model fungi cannot be readily manipulated using existing genetic tools. This project addresses this outstanding challenge by developing an integrated experimental and computational framework that enables efficient DNA delivery, precise genome editing, and scalable functional genomics across genetically diverse fungi. These capabilities will accelerate the discovery of gene functions, expand opportunities to engineer fungi for biotechnology and sustainable biomanufacturing, and advance understanding of fungal pathogens relevant to human health. By integrating experimental data with explainable artificial intelligence (AI), the project will establish predictive tools that complement experimental studies and improve the design of DNA delivery and genome engineering strategies. The project will also provide interdisciplinary research training for undergraduate and graduate students in fungal biology and biotechnology, synthetic biology, RNA biology, genome engineering, and data science. Open-source software, experimental protocols, educational resources, workshops, and peer-reviewed publications will broadly disseminate the project's tools and discoveries, thereby strengthening the nation's research infrastructure and expanding access to advanced fungal functional genomics tools. The project will establish a scalable platform for fungal functional genomics by combining advances in DNA transformation, mechanistic studies of CRISPR-based genome editing, and explainable AI models. First, the investigators will determine the molecular and physiological factors governing fungal transformability and develop a Fungal Advanced Chemical Transformation (FACT) platform by elucidating how cellular stress responses, environmental conditions, and genetic determinants regulate DNA uptake and transformation efficiency across genetically diverse fungi. Second, the investigators will elucidate the molecular mechanisms underlying CRISPR-GRIT function by investigating how gRNA biogenesis, transfer RNA-mediated processing, RNA folding, interactions with CRISPR-associated enzymes, and cellular DNA repair pathways collectively regulate genome-editing activity in fungal cells. These mechanistic studies will establish a fundamental understanding of the biological processes governing CRISPR-GRIT-mediated genome editing. Experimental measurements of RNA structure, processing, thermodynamic properties, and editing activity will then be integrated with explainable AI models to identify the molecular determinants of CRISPR-GRIT function and link molecular mechanisms to genome editing outcomes. Finally, the FACT and CRISPR-GRIT technologies will be integrated into a high-throughput CRISPR screening platform for scalable functional genomics in fungi. The platform will be used to identify genes that control important traits relevant to sustainable biomanufacturing. Together, these experimental and computational advances will establish foundational technologies and mechanistic principles that enable systematic gene-function discovery and genome engineering across genetically diverse fungi, thereby advancing biotechnology, microbial engineering, and fungal biology. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.