Mucorales are a group of early-diverging fungi with many distinct and unique features. One of the most prominent and beautiful features of some Mucorales is their response to light, producing carotenoids and showing a pronounced phototropism ^[1]. Their unusual and striking reactions to changes in the environment caught the attention of researchers in the beginnings of the first genetic studies. Among those researchers, the Nobel-awarded Max Delbrück dedicated more than 25 years studying the sensory perception in a simple cell using Phycomyces blakesleeanus as a model for more complex sensory systems. He believed that this fungus would become an essential model to develop the discipline of molecular biology and understand the interaction of an organism with its environment, the perception of information, the analysis of such information, and its corresponding responses. He also set the trend for many other researchers that continued his work. At that time, many researchers used the classic genetic methodologies (mutagenesis, phenotype selection, and mating analyses) to study the genetic regulation of these fungal responses ^[2]. However, the advances of this new mucoralean research community were soon hampered by another striking feature of Mucorales: their common reluctance to be genetically transformed ^[3]. This unconquerable disadvantage motivated many researchers to move their molecular studies to other fungal models with efficient genetic manipulation tools such as Saccharomyces, Candida, and Aspergillus [4,5]^[4][5].

The interest to study Mucorales increased at the end of the first decade of this century because of the renewed emergence of the fungal infectious disease known as mucormycosis. Mucormycosis is a lethal disease caused by several mucoralean species, being the most frequent among the genus Rhizopus, followed by Mucor and Lichtheimia (formerly Absidia) [6,7,8]^[6][7][8]. In the past, Mucormycosis was considered a rare infection related to immunosuppressed and otherwise compromised patients. However, new clinical reports and improvements in the correct diagnosis of mucormycosis have shown an emerging increase in the number of cases [9,10]^[9][10]. Indeed, the increased incidence of mucormycosis in COVID-19 patients associated with corticosteroid treatment has raised the scientific and clinical community’s concerns about treating infections caused by the so-named “black fungus” [11,12]^[11][12]. More importantly, some reports also describe an escalating number of mucormycosis cases in healthy patients without known predisposing diseases [13,14]^[13][14].

Furthermore, mucormycosis has mortality rates that can reach up to 90% in the cases of bloodstream disseminated infection [15,16]^[15][16]. These high mortality rates are mainly due to the innate antifungal drug resistance observed in Mucorales, which leaves clinicians with a few poorly effective treatments against mucormycosis [8,17,18,19,20,21]^{[8][17][18][19][20][21]}. Besides their natural high antifungal drug resistance, Mucorales can rapidly acquire new antifungal drug resistances through an exclusive RNAi-based mechanism to fast and temporally generate resistant epimutants ^[22]. In this sense, most of the current studies in Mucorales are focused on investigating new genes, pathways, methodologies, and virulence factors that might be the targets for future antifungal developments against mucormycosis [23,24,25,26,27,28,29,30,31,32]^{[23][24][25][26][27][28][29][30][31][32]}. However, this renewed interest in mucormycosis studies is still hampered by the few modern genetic tools available in Mucorales. The general reluctance of Mucorales to genetic manipulation has limited the genetic dissection of mucormycosis to the fungal model Mucor lusitanicus, previously known as Mucor circinelloides f. lusitanicus ^[33]. Homologous recombination was possible only in M. lusitanicus, which also allows other genetic tools such as genetic complementation, directed mutagenesis, and tag labeling [23,34,35,36]^{[23][34][35][36]}. 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]^[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].

2. Mucor lusitanicus, the Primary Genetic Model in Mucorales

The historical reluctance of Mucorales to genetic manipulation hampered the research of this group of fungi as model organisms. However, they are easily cultured under laboratory conditions and exhibit fast-growing and apparent phenotypes to study many biological processes. The classical model organism of Mucorales was Phycomyces blakesleeanus, to which Delbrück dedicated more than two decades studying the interaction of this model organism with the environment. Unfortunately, its inability to be transformed with exogenous DNA forced many researchers to explore other models ^[40]. The early development of an efficient transformation method of M. lusitanicus based on self-replicative plasmids ^[41] laid the foundation of this fungus as the primary genetic model in Mucorales ^[42]. This early transformation technique, based on polyethylene glycol (PEG) to allow the DNA entry into protoplast, has been refined all over the years until the successful implementation of the electroporation protocol ^[43]. Thenceforth, the genetic tools to manipulate the genome of M. lusitanicus have grown exponentially, allowing the characterization of the response to light [44^[44][45],45], RNA interference (RNAi) ^[46], pathogenesis [23^[23][32],32], lipids metabolism ^[47], carotenoids biosynthesis, centromere structure, and dimorphism [36,45,48,49,50]^{[36][45][48][49][50]}.

2.1. Plasmid Transformation, RNAi, and Functional Genomics

M. lusitanicus transformation complements auxotrophic markers such as leucine, uracil, and methionine. The obtention of auxotrophs ^[51] and the characterization of the genes that complement these phenotypes [41,52]^[41][52] allowed their use as selectable markers. Thus, the inclusion of the selectable markers in self-replicative plasmids entailed the development of the first molecular tools in M. lusitanicus. Using these selectable self-replicative plasmids as recipients to construct genomic libraries boosted the characterization of the carotenoid’s biosynthetic pathway. The filamentous fungus M. lusitanicus exhibits a yellow phenotype cultured under illumination conditions due to the accumulation of β-carotene as other Mucorales. Before discovering RNAi in M. lusitanicus, the implementation of genomic libraries and an accidentally silenced dark-yellow transformant led to the identification of a gene involved in the process of carotenogenesis, the negative regulator crgA ^[49]. This discovery led to the further complete dissection of the silencing mechanism in M. lusitanicus [26,53,54,55,56]^{[26][53][54][55][56]}.

2.2. Homologous Recombination and Its Derived Genetic Tools in M. lusitanicus

The use of self-replicative plasmids supposed the beginning of the genetic tools that placed M. lusitanicus in a unique position as a model organism. However, the true landmark in the genetics of Mucorales was the development of a methodology to edit the genome of M. lusitanicus by homologous recombination ^[49]. From gene disruption to the most recent protein-tagging strategies, the use of the homologous recombination phenomenon supposed a revolution that converted this fungus into a reference model for genetic manipulation. The first studies reporting homologous recombination in M. lusitanicus appeared right after the development of the transformation method [41,64]^[41][57]. The homologous recombination process supposes the reparation of a double-strand break (DSB) in the DNA by using a similar or identical DNA molecule to replace the region affected [65]^[58]. The observation of this process in self-replicative plasmids supposed the first hint indicating that the natural occurrence of DSB was frequent enough for applying it to develop a genome-edition method in M. lusitanicus [64,66]^[57][59].

3. Rhizopus microsporus, the New Genetic Model to Study Mucormycosis

Among all Mucorales, R. microsporus combines several characteristics that make it one of the most interesting species. This fungus is a model for studying symbiotic relationships between fungi and bacteria [86]^[60]. While some strains of R. microsporus can complete sexual and asexual reproduction independently, others require bacteria to reproduce [86]^[60]. Thus, R. microsporus is a well-known model of mutualistic symbiosis that may have evolved from a previous antagonistic interaction [87,88]^[61][62]. Moreover, this fungus is also a plant pathogen, causing rice seedling blight [89]^[63]. Symbiotic bacteria (Mycetohabitans sp., previously classified as Burkholderia) produce rhizoxin, a toxin that blocks plant mitosis and allows both fungus and bacteria to live in the necrotized plant tissue [89]^[63]. Bacterial presence and rhizoxin production are not essential for developing the lethal disease mucormycosis [90]^[64]. Infection caused by Rhizopus species supposes around 50% of all cases reported of this disease globally, being the most prevalent genus among all causal fungal agents of mucormycosis ^[14]. Along with Mucor, some mechanisms involved in the pathogenesis of mucormycosis have been unraveled in Rhizopus. For instance, macrophages are the first line of defense against the infection, and iron restriction inside the phagosomes regulates host defense [25,91]^[25][65]. In addition, the endothelial CotH proteins are a crucial element in the adhesion and invasion of the tissues ^[24].

3.1. A New R. microsporus Strain for Genetic Transformation: Auxotrophic Isolation and Plasmid Transformation

Undoubtedly, tools that allow for genetic manipulation open many possibilities. 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]. 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].

3.2. Development of a Stable Homologous Recombination Strategy Based on the CRISPR-Cas9 Machinery in R. microsporus

Although plasmid transformation is a relevant landmark itself, the main concern with self-replicative plasmid is that only a variable proportion of descendant spores will carry the plasmid through growth cycles ^[34].In addition, for applications like RNAi-induced silencing, only descendants with a high copy number of plasmid can trigger silencing mechanisms ^[34]. The benefits and possibilities that a working genetic modification procedure by homologous recombination can produce have been previously detailed with M. lusitanicus. To combine these advantages with the virulent nature of R. microsporus, efforts focused on developing an equivalent procedure in this fungus. The successful strategy comprised using in vitro assembled ribonucleoprotein complex by Cas9 and a guide RNA (gRNA) that targets a specific sequence in the genome coupled with DNA templates flanked with micro-homology repair regions (35–40 bp). These short homology regions were adapted from the previously validated strategy developed in other fungi, like Aspergillus fumigatus [92]^[66].

3.3. Uracil Auxotrophy Is Directly Related to the Virulence of R. microsporus

As a proof of concept, the wild-type R. microsporus strain, the uracil auxotrophic strain (pyrF⁻), and the pyrF complemented strains (with pyrF gene integrated either in leuA and crgA locus) were tested in mice infection experiments. In contrast with mice infection experiments with M. lusitanicus, which require strong immunosuppression and the use of specific mouse strain, R. microsporus shows an apparent virulence with immunocompetent Swiss mice ^[39]. While the wild-type strain of R. microsporus killed all mice in the first 6-7 days post-infection, the pyrF⁻ strain did not kill any mice, showing an utterly avirulent phenotype. The virulent phenotype was restored in the leuA and crgA mutant strains when they integrated a functional copy of pyrF gene ^[39]. Consistent with findings in other fungi, uracil autotrophy has also been determined as a virulence trait in A. fumigatus and Candida albicans [97,98,99]^[67][68][69].

4. Attempts to Transform Other Mucorales

4.1. Homologous Recombination in Rhizopus delemar

Rhizopus delemar (previously known as R. oryzae) is one of the most frequent causal agents isolated from patients suffering mucormycosis [100]^[70]. However, genetic manipulation in R. delemar, is quite limited. The aseptate hyphae, the multinucleated vegetative spores, and the duplicated genome are mucoralean features influencing the inefficient generation of stable null mutants [101]^[71]. The principal attempt to study a gene function in R. delemar was in the high-affinity iron uptake system by gene disruption of one of its components. Iron is an essential micronutrient for all microorganisms, and during infection, pathogenic microbes must obtain it from the host, making it an interesting target for antifungal treatments. In R. delemar, the high-affinity iron uptake system has three key elements: an iron reductase (FRE), a ferroxidase (FET3), and a permease (FTR1). A disruption approach was designed more than a decade ago using the auxotrophy marker pyrF flanked by two homology fragments for homologous recombination in the ftr1 locus. The result of this study was an unstable heterokaryon mutant, which was interpreted as evidence of the essential role of this gene in Mucorales. However, further studies demonstrated that this gene could be easily disrupted in M. lusitanicus ^[32].

4.2. CRISPR-Cas9-Based Mutagenesis in the Fungus Lichtheimia corymbifera

L. corymbifera is another causal agent of mucormycosis presenting a high isolation frequency from clinical samples right after R. delemar ^[14]. Like other Mucorales, L. corymbifera also strongly resists the traditional genetic manipulation methodologies. The lack of genetic tools has hampered the dissection of the genetic pathways behind the pathogenic potential of L. corymbifera. Homologous recombination using exogenous DNA fragments has not been achieved in L. corymbifera, not even in an unstable state like in R. delemar. However, an adapted methodology based on the CRISPR-Cas9 system and without the necessity of an autoreplicative plasmid worked in L. corymbifera to disrupt a target locus [103]^[72]. This plasmid-free system directly transformed the L. corymbifera protoplasts with the Cas9 protein and two guides RNAs (gRNA) flanking a region of the uracil selective marker gene pyrG (encoding the orotidine 5′-phosphate decarboxylase).

5. Conclusions

Regarding the pathogenesis of Mucorales, M. lusitanicus has been the primary genetic model during the last decade ^[29]. The first studies linked the size of the spore and the germination velocity with virulence ^[37]. A genomic platform based on the RNAi mechanism identified new genes involved in virulence ^[23]. The primary study of the RNAi mechanism in M. lusitanicus led to discovering an antifungal drug resistance mechanism conserved only in Mucorales based on the generation of resistant epimutants [22,26,56,68,73,74]^{[22][26][56][73][74][75]}. The high-affinity iron uptake system, an essential process in the virulence of most pathogens, was also genetically studied in M. lusitanicus ^[32]. Different genomic and transcriptomic approaches identified gene profiles related to virulence, and many genes from these profiles were mutated and functionally validated in survival assays [25,28]^[25][28]. In addition, the study of the transduction pathways in M. lusitanicus led to identifying new genes and pathways related to virulence [79,105]^[76][77]. Thus, M. lusitanicus has been an invaluable genetic model in studying genes and pathways associated with the virulence of Mucorales.

However, M. lusitanicus shows reduced virulence in the survival assays performed in the laboratory using murine models, and more striking, it has never been isolated from a patient as a causal agent of mucormycosis ^[37]. The recent development of the methodologies allowing stable homologous recombination in R. microsporus, one of the most usual causal agents of mucormycosis, represents a landmark in the study of mucormycosis. This development will make Rhizopus microsporus the leading choice for all future studies related to virulence. These recent studies showed the possibility of disrupting genes and later complementing the mutations with two different auxotrophy marker genes. The possibility of performing homologous recombination in R. microsporus predicts that other genetic techniques will soon be developed in this fungus, such as directed mutagenesis and aminoacid substitutions, overexpression, tag-labeling, and RNAi. Current and future techniques, the virulent wild type strain (positive control) and the avirulent uracil auxotrophic strain (negative control), constitute the perfect platform to study the pathogenic potential of Mucorales. Finally, the new methodology employed in transforming R. microsporus using the CRISPR-Cas technology will likely be exported to other Mucorales.