Abstract
Fungal pathogens represent a major human threat affecting more than a billion people worldwide. Invasive infections are on the rise, which is of considerable concern because they are accompanied by an escalation of antifungal resistance. Deciphering the mechanisms underlying virulence traits and drug resistance strongly relies on genetic manipulation techniques such as generating mutant strains carrying specific mutations, or gene deletions. However, these processes have often been time-consuming and cumbersome in fungi due to a number of complications, depending on the species (e.g., diploid genomes, lack of a sexual cycle, low efficiency of transformation and/or homologous recombination, lack of cloning vectors, nonconventional codon usage, and paucity of dominant selectable markers). These issues are increasingly being addressed by applying clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 mediated genetic manipulation to medically relevant fungi. Here, we summarize the state of the art of CRISPR–Cas9 applications in four major human fungal pathogen lineages: Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, and Mucorales. We highlight the different ways in which CRISPR has been customized to address the critical issues in different species, including different strategies to deliver the CRISPR–Cas9 elements, their transient or permanent expression, use of codon-optimized CAS9, and methods of marker recycling and scarless editing. Some approaches facilitate a more efficient use of homology-directed repair in fungi in which nonhomologous end joining is more commonly used to repair double-strand breaks (DSBs). Moreover, we highlight the most promising future perspectives, including gene drives, programmable base editors, and nonediting applications, some of which are currently available only in model fungi but may be adapted for future applications in pathogenic species. Finally, this review discusses how the further evolution of CRISPR technology will allow mycologists to tackle the multifaceted issue of fungal pathogenesis.
Highlights
The most common fungal diseases are superficial infections, affecting almost two billion people worldwide [1]
In this review we describe the state of the art of clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 applications in four major human fungal pathogens, Candida spp., Cryptococcus neoformans, A. fumigatus, and Mucorales, dissecting the customized strategies for the delivery of functional CRISPR–Cas9 elements
Gene manipulation in A. fumigatus is hampered by low efficiency of homology-directed repair (HDR) (3%–5%), and nonhomologous end joining (NHEJ)-impaired strains and 1-kb homology arms are traditionally used for site-specific integration [48]
Summary
The most common fungal diseases are superficial infections, affecting almost two billion people worldwide [1]. We are currently facing a concerning escalation of antifungal resistance, with reports of azole-resistant Aspergillus fumigatus and the spread of multidrug-resistant Candida auris [3,4] In this context, there is an urgent need for efficient genetic manipulation tools to further our understanding of the biology and pathophysiology of fungal pathogens and to decipher drug resistance mechanisms, which are critical for developing novel therapeutic strategies. RNA pol III promoters of some species have been characterized (e.g., SNR52 promoter in C. albicans), mining a genome to find de novo RNA pol III promoters is a cumbersome process of trial and error, which can only be partially streamlined if transcriptome data are available to guide the search For this reason, suitable RNA pol II promoters can be used instead, provided that the sgRNA molecule is flanked by RNA elements (typically ribozymes or tRNAs) that will mediate the removal of the 5’ cap and the 3’ poly(A) tail [21]. Numerous guide-design software programs are available, some allowing uploading of custom genomes, such as EUPaGDT (http://grna.ctegd. uga.edu) or CHOPCHOP (http://chopchop.cbu.uib.no/)
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