title: Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus.
abstract: Low 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.
authors: Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
authors: Kevin K
authors: KK
authors: Fuller
authors: Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
authors: Shan
authors: http://orcid.org/0000-0002-1364-4265
authors: S
authors: Chen
authors: Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA jennifer.loros@dartmouth.edu jay.c.dunlap@dartmouth.edu.
authors: Jennifer J
authors: JJ
authors: Loros
authors: Department of Genetics, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA jennifer.loros@dartmouth.edu jay.c.dunlap@dartmouth.edu.
authors: Jay C
authors: JC
authors: Dunlap
mesh_terms: D001232:Aspergillus fumigatus / Q000235:genetics*; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D005656:Fungal Proteins / Q000235:genetics; D018390:Gene Targeting / Q000379:methods; D008969:Molecular Sequence Data; D048630:Polyketide Synthases / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D048630:Polyketide Synthases