CRISPR-Cas9, the new kid on the block of fungal molecular biology

Abstract

Research on fungal pathogens with the aim to identify virulence determinants strictly relies on the generation of defined, recombinant strains, a task that is executed bymeans of a sophisticated molecular biology toolbox. Recent developments in fungal genome engineering have opened a new frontier by implementing the CRISPR-Cas9 technology, based on expression of the Cas9 endonuclease that is loaded by a single guiding RNA (sgRNA) molecule to target a defined site in the recipient genome. This novel approach has been adapted successfully to engineer fungal genomes, among them the one of the human-pathogenic mould Aspergillus fumigatus. Implementation of the required components was achieved by various means that differ with respect to expression of the Cas9 enzyme and sgRNA delivery. Validation of CRISPR-Cas9-mediated mutagenesis could be executed by targeting selected candidate genes of A. fumigatus to provide a promising perspective for screening and multiplexing approaches to scrutinize the virulome of this opportunistic fungal pathogen in a comprehensive manner, such as by analyzing genetic polymorphisms or the function of gene families.