The CRISPR toolbox in medical mycology: State of the art and perspectives
Résumé
AbstractFungal pathogens represent a major human threat affecting more than a billion peopleworldwide. Invasive infections are on the rise, which is of considerable concern becausethey are accompanied by an escalation of antifungal resistance. Deciphering the mecha-nisms underlying virulence traits and drug resistance strongly relies on genetic manipulationtechniques such as generating mutant strains carrying specific mutations, or gene deletions.However, these processes have often been time-consuming and cumbersome in fungi dueto a number of complications, depending on the species (e.g., diploid genomes, lack of asexual cycle, low efficiency of transformation and/or homologous recombination, lack ofcloning vectors, nonconventional codon usage, and paucity of dominant selectable mark-ers). These issues are increasingly being addressed by applying clustered regularly inter-spaced short palindromic repeats (CRISPR)–Cas9 mediated genetic manipulation tomedically relevant fungi. Here, we summarize the state of the art of CRISPR–Cas9 applica-tions in four major human fungal pathogen lineages: Candida spp., Cryptococcus neofor-mans, Aspergillus fumigatus, and Mucorales. We highlight the different ways in whichCRISPR has been customized to address the critical issues in different species, includingdifferent strategies to deliver the CRISPR–Cas9 elements, their transient or permanentexpression, use of codon-optimized CAS9, and methods of marker recycling and scarlessediting. Some approaches facilitate a more efficient use of homology-directed repair in fungiin which nonhomologous end joining is more commonly used to repair double-strand breaks(DSBs). Moreover, we highlight the most promising future perspectives, including genedrives, programmable base editors, and nonediting applications, some of which are cur-rently available only in model fungi but may be adapted for future applications in pathogenicspecies. Finally, this review discusses how the further evolution of CRISPR technology willallow mycologists to tackle the multifaceted issue of fungal pathogenesis.
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