Li Yuwen, Dai Jing, Zhang Huan, Zhang Han, Guzman Adrian, Han Song-I, Shim Won Bo, Han Arum
Department of Electrical and Computer Engineering, Texas A&M University, TX, USA.
Department of Plant Pathology and Microbiology, Texas A&M, University, TX, USA.
Anal Chim Acta. 2025 Jul 8;1358:344094. doi: 10.1016/j.aca.2025.344094. Epub 2025 Apr 18.
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.
真菌是一类多样的真核生物，全球生态系统中有超过150万种真菌。许多真菌以丝状（菌丝）形式生长，发挥着至关重要的生态作用，既有有益的，也有有害的。了解它们的功能通常需要生成基因敲除突变体并与野生型菌株进行比较分析。然而，传统的筛选基因敲除突变体的方法劳动强度大、耗时，并且限制了对成功转化体的快速鉴定。在此，我们展示了一个高通量液滴微流控平台，该平台能够以单细胞分辨率筛选和分选真菌转化体，与传统方法相比，显著提高了效率。工作流程包括将单个真菌转化体封装在皮升体积的油包水乳液液滴中，在抗生素存在的情况下培养它们，识别并分选含有表现出菌丝生长的转化体的液滴。在抗生素存在下生长的转化体被标记为潜在的基因敲除体，然后分选出来通过测序进行确认。这种方法具有几个优点，包括将禾谷镰刀菌原生质体生长到可以与不生长的原生质体区分开来的时间减少了3倍，每小时高达28,800个含转化体液滴的筛选通量，以及单孢子表型分析以尽量减少后处理要求。使用这个系统，我们成功筛选了24,000个含液滴的禾谷镰刀菌转化体，鉴定出五个在琼脂平板上生长的潜在转化体，其中两个通过测序确认为真正的基因敲除体。这些结果证明了这个基于液滴微流控的平台作为加速真菌功能基因组学和推进我们对真菌生态作用理解的强大工具的实用性。
