Assembly of Markerless CRISPR-Cas9 Deletion Construction in Candida auris

Abstract

Candida auris is an emerging multi-drug resistant fungal pathogen causing grave concern. In Candida auris, a gene orthologous to the YAP1 gene in Saccharomyces cerevisiae is present. We suspect the YAP1 orthologous gene to have a similar function to that of S. cerevisiae, in which it is responsible for the oxidative stress response. In order to delete the gene and examine its function, a deletion construction employing CRISPR-Cas9 was assembled. CRISPR-Cas9 is a simple and flexible gene editing technology that is capable of cutting DNA at a specific locus. We designed and extracted fragments comprising of the CRISPR component, single guide RNAs, temporary selective marker nourseothricin, and leucine gene to excise the gene.