1. Abstract Rhizopus delemar is the leading casual-agent of mucormycosis, a life-threatening fungal infection in immunocompromised patients. Despite its clinical importance, R. delemar remains understudied due to its limited genetic tractability and lack of available research resources. Specifically, R. delemar produces coenocytic hyphae and multinucleated spores, resulting in a high difficulty obtaining stable gene-specific mutant strains. We have previously employed consecutive hyphal tip microdissection, and with CRISPR-Cas9 techniques, to isolate mononuclear mutant strains. However, this isolation method is laborious and inefficient. To circumvent this obstacle, we propose hyphal fragmentation and protoplast generation, coupled with fluorescence-activated cell sorting (FACS), which allows isolation of mononuclear propagules to obtain mutant clones following genetic transformation robustly. We will first optimize hyphal fragmentation and protoplast production to enrich single nucleated cells (Aim 1a) and label R. delemar strains with fluorescent nuclear markers through DNA transformation (Aim 1b). We will then sort and isolate mononuclear hyphal fragments/protoplasts through FACS analysis (Aim 2a). Additionally, we will improve the DNA plasmid for further genetic manipulation of R. delemar (Aim 2b). Our research plan's successful implementation will provide an invaluable tool to facilitate genetic studies of R. delemar-induced mucormycosis mechanisms.