The CRISPR toolbox in medical mycology: State of the art and perspectives
Résumé
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 mecha-
nisms 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 mark-
ers). These issues are increasingly being addressed by applying clustered regularly inter-
spaced short palindromic repeats (CRISPR)–Cas9 mediated genetic manipulation to
medically 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 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 cur-
rently 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.
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