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 which are required for M. circinelloides to resist antifungal agents and establish a
foundation for the development of therapeutic strategies to overcome mucormycosis.
