Transformation and CRISPR-Cas9-mediated homologous recombination in the fungus Rhizopus microsporus

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

doi:10.1016/j.xpro.2022.101237

Cited by 3 publications

(5 citation statements)

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“…In this sense, the dissection of mucoralean pathogenic potential must also include approaches designed to find new virulence factors undescribed in other pathogens. Whole-genome sequencing of several Mucorales species has revealed a significant number of “unknown function genes” that may play a critical role in their virulence [ 103 , 107 ]. The study of an unknown function gene in Mucorales represents a challenge with more complexity than a gene with information previously obtained in other organisms.…”

Section: Discussionmentioning

confidence: 99%

“…However, it is worth noting that M. lusitanicus has shown reduced virulence in laboratory survival assays using murine models and has never been isolated as a causing agent of mucormycosis. The recent development of methodologies allowing stable homologous recombination in R. microsporus , one of the most common causing agents of mucormycosis, represents a significant breakthrough in the study of mucormycosis [ 9 , 103 ]. This development will make R. microsporus the preferred choice for future studies related to virulence.…”

Section: Discussionmentioning

confidence: 99%

“…Although the features described above point to R. microsporus as the perfect model to study virulence and mucormycosis, its reluctance to genetic manipulation delegated these studies to the less virulent fungus M. lusitanicus . Fortunately, recent work has successfully developed a CRISPR-Cas9 plasmid-free methodology for the genetic manipulation of R. microsporus [ 9 , 103 ]. This work designed a platform for virulence studies in R. microsporus that included a specifically selected initial strain and positive and negative controls, besides the optimized methodology for successful homologous recombination.…”

Section: Genetic Manipulation In Mucorales and New...mentioning

confidence: 99%

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Mucorales and Mucormycosis: Recent Insights and Future Prospects

Tahiri

Lax

Cánovas-Márquez

et al. 2023

JoF

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The classification of Mucorales encompasses a collection of basal fungi that have traditionally demonstrated an aversion to modern genetic manipulation techniques. This aversion led to a scarcity of knowledge regarding their biology compared to other fungal groups. However, the emergence of mucormycosis, a fungal disease caused by Mucorales, has attracted the attention of the clinical field, mainly because available therapies are ineffective for decreasing the fatal outcome associated with the disease. This revitalized curiosity about Mucorales and mucormycosis, also encouraged by the recent COVID-19 pandemic, has spurred a significant and productive effort to uncover their mysteries in recent years. Here, we elaborate on the most remarkable breakthroughs related to the recently discovered genetic advances in Mucorales and mucormycosis. The utilization of a few genetic study models has enabled the identification of virulence factors in Mucorales that were previously described in other pathogens. More notably, recent investigations have identified novel genes and mechanisms controlling the pathogenic potential of Mucorales and their interactions with the host, providing fresh avenues to devise new strategies against mucormycosis. Finally, new study models are allowing virulence studies that were previously hampered in Mucorales, predicting a prolific future for the field.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Genetic Manipulation In Mucorales and New...mentioning

confidence: 99%

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Mucorales and Mucormycosis: Recent Insights and Future Prospects

Tahiri

Lax

Cánovas-Márquez

et al. 2023

JoF

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“…Consistent with CRISPR-Cas9- induced gene mutation by non-homologous end joining (NHEJ) the CRISPR/Cas9 has been utilized to create R. delemar pyrF mutants with a single nucleotide deletion at the fourth nucleotide before the protospacer adjacent motif (PAM) sequence ( Bruni et al., 2019 ). The CRISPR-Cas9 targeted integration method was also recently adapted to reliably and stably transform protoplasts of R. microsporus ( Lax et al., 2021 ; Lax et al., 2022 ). In M. lusitanicus , the CRISPR-Cas9 system has been successfully employed in a plasmid-free way to disrupt two genes: carB, which encodes phytoene dehydrogenase, and hmgR2, which encodes 3-hydroxy-3-methylglutaryl-CoA reductase ( Nagy et al., 2017 ).…”

Section: Genomic Structure and Genetic Manipulation To Understand Pat...mentioning

confidence: 99%

Mucormycosis in 2023: an update on pathogenesis and management

Alqarihi,

Kontoyiannis,

Ibrahim

2023

Front. Cell. Infect. Microbiol.

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Mucormycosis (MCR) is an emerging and frequently lethal fungal infection caused by the Mucorales family, with Rhizopus, Mucor, and Lichtheimia, accounting for > 90% of all cases. MCR is seen in patients with severe immunosuppression such as those with hematologic malignancy or transplantation, Diabetes Mellitus (DM) and diabetic ketoacidosis (DKA) and immunocompetent patients with severe wounds. The recent SARS COV2 epidemy in India has resulted in a tremendous increase in MCR cases, typically seen in the setting of uncontrolled DM and corticosteroid use. In addition to the diversity of affected hosts, MCR has pleiotropic clinical presentations, with rhino-orbital/rhino-cerebral, sino-pulmonary and necrotizing cutaneous forms being the predominant manifestations. Major insights in MCR pathogenesis have brought into focus the host receptors (GRP78) and signaling pathways (EGFR activation cascade) as well as the adhesins used by Mucorales for invasion. Furthermore, studies have expanded on the importance of iron availability and the complex regulation of iron homeostasis, as well as the pivotal role of mycotoxins as key factors for tissue invasion. The molecular toolbox to study Mucorales pathogenesis remains underdeveloped, but promise is brought by RNAi and CRISPR/Cas9 approaches. Important recent advancements have been made in early, culture-independent molecular diagnosis of MCR. However, development of new potent antifungals against Mucorales remains an unmet need. Therapy of MCR is multidisciplinary and requires a high index of suspicion for initiation of early Mucorales-active antifungals. Reversal of underlying immunosuppression, if feasible, rapid DKA correction and in selected patients, surgical debulking are crucial for improved outcomes.

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“…В то же время Mucor может быть комменсалом носовой полости здоровых субъектов [6]. Геномный анализ Mucorales, изолированных до пандемии COVID-19, вместе с исследованиями на экспериментальных животных может свидетельствовать о возможной роли более вирулентных штаммов в возникновении СММ [7]. Тем не менее, в отличие от возбудителя COVID-19 SARS-CoV-2, постоянно подверженного мутациям, структура грибов Mucorales в основном стабильна, хотя и описаны некоторые геноспецифические различия во взаимодействии их с иммунными клетками человека [8].…”

Section: Introductionunclassified

COVID-19 and mucormycosis

Lipnitsky¹,

Polovets²,

Surkova³

et al. 2021

Immunopathol Allergol Infectol

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Mucormycosis is an angioinvasive disease caused by fungi of the order Mucorales. Coronavirus disease (COVID-19), associated mucormycosis (CAM) is a global emerging threat. More than 40 000 CAM cases have been reported in India. Emergense of CAM has been attributed to host, environmental and agents-related factors. Mucorales spore burden has been reported globally, however their presence is higher in tropical countries, such as India. Before the COVID-19 pandemic, patients with diabetes mellitus, hematological malignancies, solid organ transplants, corticosteroid therapy and neutropenia were more prone to mucormycosis. In COVID-19 patients, virus-induced endothelial dysfunction, hyperglycemia, and immune dysfunction following corticosteroid use. COVID-19 infection may directly induce hyperglycaemia by damaging beta cells of the pancreas or by corticosteroid therapy. In addition, corticosteroid treatment reduces the innate immune function of phagocytic cells. Free iron acquisition from the host is an important virulence factor of Mucorales.

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Transformation and CRISPR-Cas9-mediated homologous recombination in the fungus Rhizopus microsporus

Cited by 3 publications

References 11 publications

Mucorales and Mucormycosis: Recent Insights and Future Prospects

Mucorales and Mucormycosis: Recent Insights and Future Prospects

Mucormycosis in 2023: an update on pathogenesis and management

COVID-19 and mucormycosis

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