Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

Abstract

bLow rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen.

Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering

“…The functional CRISPR/Cas9 system for gene editing has been successfully developed in yeasts and some fungi [28–40], including industrial filamentous fungi Aspergillus stains [33, 40], Trichoderma reesei [34] and Penicillium chrysogenum [36], and plant pathogenic fungi A. fumigatus [35, 38], Magnaporthe oryzae [37], and Ustilago maydis [39]. For instance, by using the CRISPR/Cas9 technology in T. reesei [34], simultaneous engineering of multiple genes was efficiently achieved through co-transformation with in vitro synthesized gRNAs and donor DNA even using short 200-bp homology arms, providing an applicable and promising approach to other lignocellulose degrading filamentous fungi.…”

CRISPR/Cas9-mediated efficient genome editing via blastospore-based transformation in entomopathogenic fungus Beauveria bassiana

“…The sgRNA can recruit Cas9 to create a specific DNA double strand break (DSB) in the target region, leading to deletions, insertions and substitutions through error-prone NHEJ in the absence of a homologous sequence or gene specific replacement through homologous recombination-directed repair (HDR) in the presence of a homologous DNA repair template (donor DNA)2732. The CRISPR/Cas9 system has been adapted to several fungi, such as yeasts Schizosaccharomyces pombe 33, S. cerevisiae 34, filamentous ascomycete fungi Trichoderma reesei 35, Neurospora crassa 36, M. oryzae 37 and Aspergillus fumigatus 383940. However, the CRISPR/Cas9 system or other genome-editing approaches have not yet been reported in entomopathogenic fungi.…”

CRISPR/Cas9-The ultimate weapon to battle infectious diseases?

“…Systemic fungal infections caused by the filamentous fungus Aspergillus species and the yeast species Cryptococcus and Candida have markedly risen over the past decade, due to an increase in both the aged population and the number of immune‐compromised individuals, as well as antifungal‐resistant subspecies (Pfaller & Diekema, ). The CRISPR/Cas9 system has been recently applied in genome editing of Candida albicans (Min, Ichikawa, Woolford, & Mitchell, ; Vyas et al, ), Aspergillus (Fuller, Chen, Loros, & Dunlap, ), and Cryptococcus (Wang, Wei et al, ) and has the potential to further uncover the molecular basis of fungal infection and resistance to antifungals (Table ).…”