ABSTRACT
In this chapter, the authors describe the state of the art of CRISPR-Cas9 applications in four major human fungal pathogens, Candida spp., Cryptococcus neoformans, A. fumigatus, and Mucorales, dissecting the customized strategies for the delivery of functional CRISPR-Cas9 elements. Expression of CAS9, codon optimization, and nuclear localization of the protein are crucial for the success of CRISPR-Cas9 gene editing. In addition, the expression of a sgRNA suitable for interaction with Cas9 is pivotal. CRISPR-Cas9 methods have also been implemented in non-albicans Candida species. Like C. albicans, the C. parapsilosis sensu lato complex species, C tropicalis, C. lusitaniae, and C. auris are members of the CTG clade. CRISPR-Cas9 made the construction of genome-scale mutant libraries easier than ever before, thus streamlining downstream applications such as genomewide variant engineering or gene interaction screens. The use of RNA-Cas9 complexes is particularly important in species refractory to genetic manipulation, making easier and more timely the study of emergent and multiresistant fungi.
