Stable and reproducible homologous recombination enables CRISPR-based engineering in the fungus Rhizopus microsporus

Lax, Carlos; Navarro-Mendoza, María Isabel; Pérez-Arques, Carlos; Navarro, Eusebio; Nicolás, Francisco E.

doi:10.1016/j.crmeth.2021.100124

Cited by 21 publications

(31 citation statements)

References 79 publications

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“…Similar approaches have been followed in R. delemar and L. corymbifera , but in these species, the procedures require optimization because they work exclusively in genes that produce a selectable phenotype after mutation ( Bruni et al., 2019 ; Ibragimova et al., 2020 ), making unfeasible their widespread use. In this context, a promising plasmid-free CRISPR-Cas9-based procedure has been established for R. microsporus that produces stable transformants, allows targeted mutation of any gene, and uses microhomology repair templates, speeding up the whole process ( Lax et al., 2021 ).…”

Section: New Genetic Tools To Study Gene Functionmentioning

confidence: 99%

Recent Advances and Future Directions in the Understanding of Mucormycosis

Garre

2022

Front. Cell. Infect. Microbiol.

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Mucormycosis is an emerging infection caused by fungi of the order Mucorales that has recently gained public relevance due to the high incidence among COVID-19 patients in some countries. The reduced knowledge about Mucorales pathogenesis is due, in large part, to the historically low interest for these fungi fostered by their reluctance to be genetically manipulated. The recent introduction of more tractable genetic models together with an increasing number of available whole genome sequences and genomic analyses have improved our understanding of Mucorales biology and mucormycosis in the last ten years. This review summarizes the most significant advances in diagnosis, understanding of the innate and acquired resistance to antifungals, identification of new virulence factors and molecular mechanisms involved in the infection. The increased awareness about the disease and the recent successful genetic manipulation of previous intractable fungal models using CRISPR-Cas9 technology are expected to fuel the characterization of Mucorales pathogenesis, facilitating the development of effective treatments to fight this deadly infection.

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Section: New Genetic Tools To Study Gene Functionmentioning

confidence: 99%

Recent Advances and Future Directions in the Understanding of Mucormycosis

Garre

2022

Front. Cell. Infect. Microbiol.

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“…Given the limitations and the difficulties associated with genetic modification of Mucorales, a step-by-step optimization of the process was necessary to achieve this complex and longed-for goal. Analogously to previous studies, a uracil auxotrophic strain of R. microsporus (ATCC 11559) was isolated [ 39 ]. However, unlike previous approaches, the R. microsporus auxotrophic strain was isolated spontaneously without mutagenic compounds or UV light [ 39 ].…”

Section: Rhizopus Microsporus the New Genetic Model To Stud...mentioning

confidence: 99%

“…Analogously to previous studies, a uracil auxotrophic strain of R. microsporus (ATCC 11559) was isolated [ 39 ]. However, unlike previous approaches, the R. microsporus auxotrophic strain was isolated spontaneously without mutagenic compounds or UV light [ 39 ]. This reduced mutational burden is desirable considering that this strain (UM1) will be used for downstream analysis and characterization, especially virulence assays.…”

Section: Rhizopus Microsporus the New Genetic Model To Stud...mentioning

confidence: 99%

“…This reduced mutational burden is desirable considering that this strain (UM1) will be used for downstream analysis and characterization, especially virulence assays. This strain carries a non-synonymous substitution in the pyrF gene that changes a lysine residue in the active center for a glutamic acid residue (K73E) [ 39 ].…”

Section: Rhizopus Microsporus the New Genetic Model To Stud...mentioning

confidence: 99%

“…These methodologies were used to dissect several genetic mechanisms in Mucorales, including the light responses, the RNAi mechanism, and more recently, the virulence of Mucorales. However, M. lusitanicus is not virulent without a strongly immunosuppressed host and an unnaturally high dose of spores in the initial injection [ 37 , 38 ] limitations to genetic studies on pathogenic Mucorales have been recently overcome with a new methodology to transform the fungus Rhizopus microsporus , an actual mucormycosis agent frequently isolated from patients [ 39 ]. This new genetic model represents a landmark in the current study of mucormycosis and will likely become the leading model in future studies.…”

Section: Introductionmentioning

confidence: 99%

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Genetic Manipulation in Mucorales and New Developments to Study Mucormycosis

Lax

Cánovas-Márquez

Tahiri

et al. 2022

IJMS

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The study of the Mucoralean fungi physiology is a neglected field that the lack of effective genetic tools has hampered in the past. However, the emerging fungal infection caused by these fungi, known as mucormycosis, has prompted many researchers to study the pathogenic potential of Mucorales. The main reasons for this current attraction to study mucormycosis are its high lethality, the lack of effective antifungal drugs, and its recent increased incidence. The most contemporary example of the emergence character of mucormycosis is the epidemics declared in several Asian countries as a direct consequence of the COVID-19 pandemic. Fortunately, this pressure to understand mucormycosis and develop new treatment strategies has encouraged the blossoming of new genetic techniques and methodologies. This review describes the history of genetic manipulation in Mucorales, highlighting the development of methods and how they allowed the main genetic studies in these fungi. Moreover, we have emphasized the recent development of new genetic models to study mucormycosis, a landmark in the field that will configure future research related to this disease.

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Recent Advances and Future Perspectives in Mitigating Invasive Antifungal-Resistant Pathogen Aspergillus fumigatus in Africa

Achilonu,

Davies,

Kanu

et al. 2023

Curr Treat Options Infect Dis

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Purpose of Review Azole resistance in Aspergillus fumigatus is an emerging public health issue with global distribution and has been linked to use in agricultural and horticultural settings. In 2022, the World Health Organization (WHO) created a fungal pathogen priority list, and A. fumigatus was listed as a critical pathogen. Currently, Africa lacks effective surveillance systems for this emerging threat, mostly due to lack of capacity and diagnostics to determine azole resistance in routine clinical settings. This review aims to address and improve on the current diagnostic tools and future perspective strategies in tackling clinical and environmental antifungal-resistant (AFR) A. fumigatus in Africa. We emphasized on the importance of early diagnosis and misdiagnosis associated with aspergillosis caused by Aspergillus sp., cross talk between clinical and environmental, mode of action and resistance mechanism, collaborative one health approach, and future perspectives for AFR A. fumigatus management strategies. Recent Findings Early diagnosis and effective management of invasive aspergillosis are critical. On the continent, very few laboratories routinely conduct antifungal susceptibility testing on Aspergillus species. Where this occurs, it is culture-based in vitro antifungal susceptibility testing. Drug repurposing and the need for a non-culture-based molecular method (PCR) are critical. Summary Enhancing promising future perspectives of non-cultured approaches such as whole-genome sequencing, CRISPR/Cas9, and RNAi-mediated technologies to complement the culture-based approach as important strategies to mitigate and overcome emerging issues of AFR A. fumigatus in Africa.

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Stable and reproducible homologous recombination enables CRISPR-based engineering in the fungus Rhizopus microsporus

Cited by 21 publications

References 79 publications

Recent Advances and Future Directions in the Understanding of Mucormycosis

Recent Advances and Future Directions in the Understanding of Mucormycosis

Genetic Manipulation in Mucorales and New Developments to Study Mucormycosis

Recent Advances and Future Perspectives in Mitigating Invasive Antifungal-Resistant Pathogen Aspergillus fumigatus in Africa

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