A CRISPR/Cas9-Based Strategy to Simultaneously Inactivate the Entire <i>ALS</i> Gene Family in <i>Candida Orthopsilosis</i>

Abstract

<b>Aim:</b> In this study, the CRISPR gene-editing approach was used to simultaneously inactivate all three members of the <i>ALS</i> gene family in the opportunistic pathogen <i>Candida orthopsilosis</i>. <b>Materials & methods:</b> Using a single gRNA and repair template, CRISPR-edited clones were successfully generated in a one-step process in both <i>C. orthopsilosis</i> reference and clinical strains. <b>Results:</b> The phenotypic characterization of the <i>ALS</i> triple-edited strains revealed no impact on growth in liquid or solid media. However, pseudohyphal formation and the ability to adhere to human buccal epithelial cells were significantly decreased in triple-edited clones. <b>Conclusion:</b> Our CRISPR/Cas9 system is a powerful tool for simultaneous editing of fungal gene families, which greatly accelerates the generation of multiple gene-edited <i>Candida</i> strains. <b>Data deposition:</b> Nucleotide sequence data are available in the GenBank databases under the accession numbers MK875971, MK875972, MK875973, MK875974, MK875975, MK875976, MK875977.