Location, location, location: Use of CRISPR-Cas9 for genome editing in human pathogenic fungi

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 systems enable the targeting of a double-strand break in genomic DNA to a location chosen by the investigator [1].The break may be repaired (Fig 1) by cellular nonhomologous end joining (NHEJ) machinery to yield small insertions and deletions [2], which are often loss-of-function mutations in coding regions.If a repair template that spans the break site is available, the break may be repaired by cellular homology-dependent repair (HR) machinery to create a precisely designed insertion, deletion, gene or promoter fusion, or base substitution [2].CRISPR-Cas9 systems can be programmed to define the location of a break; hence, an investigator can determine the location of a targeted mutation (Fig 1).This technology has revolutionized genetic manipulation of model organisms, cell lines, animals, plants, and even human embryos [1].The technology has been adapted for use in a variety of fungi (reviewed in [3]), including Saccharomyces cerevisiae [4] and multiple Aspergillus species of agricultural and industrial importance [5].Among human fungal pathogens, CRISPR-Cas9 genome editing has been applied successfully to Aspergillus fumigatus [6,7], Cryptococcus neoformans var.neoformans [8], C. neoformans var.grubii [9], Candida albicans [10,11], and Ca.glabrata [12].In these organisms, CRISPR-Cas9 systems have accelerated the creation of gene-targeted mutations, often without selection for the mutant phenotype, and have enabled the modification of multiple genes or alleles in a single transformation.