PROJECT SUMMARY Mucor circinelloides is a Mucoralean mold and a primary causative pathogen of mucormycosis, a severe and life-threatening infection among immunocompromised patient populations. Unfortunately, M. circinelloides possesses a high degree of intrinsic resistance to antifungals, leaving clinicians with few options when treating patients with mucormycosis. A critical barrier to the development of strategies to overcome M. circinelloides infections is a current lack of available genetic tools and a consequent lack of understanding of the genetic and molecular basis of its ability to resist antifungals. Our long-term goal is to advance the treatment of invasive fungal infections by developing new therapeutic strategies to overcome difficult-to-treat fungal pathogens. The overall objective of this proposal is to develop a recyclable and Mucor circinelloides-optimized Cas9 gene-drive system capable of multi-target gene-editing, and to implement the M-Drive system to identify and characterize the specific M. circinelloides efflux pump-encoding genes which impact M. circinelloides antifungal susceptibility and represent novel genetic and molecular weak-points which can be therapeutically exploited for the development of future antifungal therapies. To achieve this, we will test our central hypothesis that 1) that the multinucleate nature of M. circinelloides cells can be exploited to implement an efficient and recyclable Cas9- mediated gene-drive system, 2) disruption of specific M. circinelloides efflux pump genes which are responsive to posaconazole treatment will have an impact on M. circinelloides antifungal susceptibility, and 3) leveraging the M-Drive system will allow for the rapid identification and characterization of these M. circinelloides efflux pump-encoding genes which influence antifungal susceptibility. In Aim 1, we will utilize our M. circinelloides pyrG- marker system to construct the M-Drive compatible (Cas9+) strain and confirm the functionality and efficiency of the M-Drive system by disrupting the previously characterized carRP and cnbR genes simultaneously. In Aim 2, we will implement the M-Drive system to disrupt a prioritized set of efflux pump- encoding genes which are transcriptionally up-regulated in response to posaconazole treatment and determine their impact on susceptibility to posaconazole as well as other antifungal agents. Our approach is innovative and contains important technical and conceptual advances that are expected to contribute significantly to the study of mucormycosis and to have a positive impact on the understanding of the genetic liabilities of M. circinelloides. The proposed research is significant in that upon completion, we will have created Mucor- optimized genetic tools will make possible, for the first time, rapid and efficient gene-editing in this model Mucoralean pathogen. These studies will then accelerate the comprehensive characterization of M. circinelloides genes and gene families w...