Rapid filamentous fungi gene knockout identification through high-throughput droplet microfluidics.

Fungi are a diverse group of eukaryotic organisms, with over 1.5 million species inhabiting ecosystems worldwide. Many fungi grow as filaments (hyphae) and play critical ecological roles, both beneficial and harmful. Understanding their functions often requires generating knockout mutants and performing comparative analyses with wild-type strains. However, traditional methods for screening knockout mutants are labor-intensive, time-consuming, and limit the rapid identification of successful transformants. Here, we present a high-throughput droplet microfluidics platform capable of screening and sorting fungal transformants at single-cell resolution, significantly improving efficiency compared to conventional methods. The workflow involves encapsulating individual fungal transformants in pico-liter-volume water-in-oil emulsion droplets, culturing them in the presence of antibiotics, and identifying and sorting droplets containing transformants that exhibit hyphal growth. Transformants that grow in the presence of antibiotics are flagged as potential knockouts and then sorted out for confirmation through sequencing. This approach offers several advantages, including a 3-fold reduction in time for Fusarium graminearum protoplast growth until they can be distinguished from those exhibiting no growth, a screening throughput of up to 28,800 transformant-containing droplets per hour, and single-spore phenotyping to minimize post-processing requirements. Using this system, we successfully screened 24,000 F. graminearum transformants containing droplets, identified five potential transformants that exhibit growth on agar plates, of which two were confirmed via sequencing as true knockouts. These results demonstrate the utility of this droplet microfluidics-based platform as a powerful tool for accelerating fungal functional genomics and advancing our understanding of the ecological roles of fungi.