Plasmid-Based CRISPR–Cas9 Editing in Multiple Candida Species

CRISPR-Cas9 technology radically changed the approach to genetic manipulation of both medically and industrially relevant Candida species, as attested by the ever-increasing number of applications to the study of pathogenesis, drug resistance, gene expression, and host pathogen interaction and drug discovery. Here, we describe the use of plasmid-based systems for high efficiency CRISPR-Cas9 gene editing into any strain of four non-albicans Candida species, namely, Candida parapsilosis, Candida orthopsilosis, Candida metapsilosis, and Candida tropicalis. The plasmids pCP-tRNA and pCT-tRNA contain all the elements necessary for expressing the CRISPR-Cas9 machinery, and they can be used in combination with a repair template for disrupting gene function by insertion of a premature stop codon or by gene deletion. The plasmids are easily lost in the absence of selection, allowing scarless gene editing and minimizing detrimental effects of prolonged Cas9 expression.