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Principal Investigator: Jeffrey M. Rybak
Organization: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
Fiscal Year: 2024
Award: $83,000
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Terms: <A fumigatus><A. fumigatus><Acceleration><Address><Affect><Antifungal Therapy><Applied Research><Applied Science><Aspergillus fumigatus><Azoles><Basic Research><Basic Science><C albicans><C. albicans><C.albicans><CNBr><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Candida albicans><Cas nuclease technology><Causality><Cell Body><Cell Nucleus><Cells><Characteristics><Clinical><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Communicable Diseases><Cyanogen Bromide><Cyanogen bromide (Br)><DNA cassette><Death Rate><Development><Diabetes Mellitus><Etiology><Filamentous Fungi><Foundations><Fungal Drug Resistance><Fungus Diseases><Fungus drug resistant><Future><Gene Family><Gene Transcription><Genes><Genetic><Genetic Markers><Genetic Transcription><Genome><Goals><Guide RNA><Immunocompromised><Immunocompromised Host><Immunocompromised Patient><Immunosuppressed Host><Infection><Infectious Disease Pathway><Infectious Diseases><Infectious Disorder><Investigation><Knowledge><Life><Mediating><Mission><Modeling><Modernization><Molds><Molecular><Morphology><Mucor><Mucormycosis><Mycoses><NIAID><National Institute of Allergy and Infectious Disease><Nature><Nucleus><Organism><Ortholog><Orthologous Gene><Outcome><Patients><Phenotype><Population><Predisposition><Public Health><RNA Expression><Reproduction spores><Research><Septate><Spores><Susceptibility><System><Testing><Therapeutic><Therapeutic Fungicides><Time><Transcription><Work><Zygomycosis><anti-fungal><anti-fungal agents><anti-fungal drug><anti-fungal drug resistance><anti-fungal drug resistant><anti-fungal resistance><anti-fungal resistant><anti-microbial resistant pathogen><antimicrobial resistant pathogen><causation><developmental><diabetes><disease causation><efflux pump><enhancer cassette><expression cassette><fungal infection><fungal infectious disease treatment><fungal pathogen><fungi pathogen><fungus><fungus drug resistance><fungus infection><gRNA><gene biomarker><gene cassette><gene drive approach><gene drive strategy><gene drive system><gene drive technology><gene editing platform><gene editing system><gene editing technology><gene editing tools><gene expression biomarker><gene manipulation><gene marker><gene signature biomarker><gene-editing toolkit><genetic biomarker><genetic cassette><genetic manipulation><genetic technology><genetically manipulate><genetically perturb><immunosuppressed patient><innovate><innovation><innovative><integration cassette><living system><model organism><mortality rate><mortality ratio><new approaches><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapy approaches><new treatment approach><new treatment strategy><next-generation gene drive><novel><novel approaches><novel strategies><novel strategy><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapy approach><paralog><paralogous gene><pathogen><pathogenic fungus><patient population><posaconazole><preservation><prevent><preventing><promoter cassette><reporter cassette><resistance cassette><resistance to anti-fungal><resistant to anti-fungal><response><selectable cassette><selection cassette><stop cassette><therapeutic agent development><therapeutic development><tool><transcription cassette><transcriptional cassette><transgene cassette>