• CRISPR-Cas9
• Candida orthopsilosis
• ALS
• adhesins
• mutant strain
• adhesion
• HBEC
• triple mutant

Candida orthopsilosis is an opportunistic fungal human pathogen responsible for mucosal and systemic infections that can be fatal in immunocompromised individuals. Virulence of opportunistic fungal pathogens is a multifactorial process, encompassing adhesion to biotic and abiotic substrates, morphological switching from yeast cells to pseudohyphae, biofilm formation and secretion of hydrolytic enzymes. Among these, adhesion represents the first event occurring in the onset of candidiasis. One of the most extensively studied group of adhesins are encoded by the Agglutinin-Like Sequence gene (ALS) family. Despite their broadly acknowledged importance as virulence factors, no information concerning the biological role of the Als proteins in other clinically relevant Candida species such as C. orthopsilosis is currently available. The recent sequencing and annotation of C. orthopsilosis genome (strain 90-125) allowed the identification of three putative ALS-like genes, namely CORT0B00800, CORT0C04210, and CORT0C04220 (further referred as ALS800, ALS4210, ALS4220). The role of each ALS gene has been investigated in Candida albicans through the creation of single knockout strains. However, this approach can be time-consuming, especially when dealing with multi-gene families, where simultaneous mutations at multiple loci are required. In 2015, the first CRISPR-Cas9 system in C. albicans was developed at the Massachusetts Institute of Technology (MIT, Boston, USA) using an integrative cassette. Notwithstanding, the CaCAS9 (codon optimized C. albicans Cas9) gene remained integrated in the genome which could lead to off-target effects of the endonuclease activity. To circumvent this issue, in 2017 a plasmid-based CRISPR-Cas9 system was developed in Candida parapsilosis at the University College of Dublin (UCD, Dublin, Ireland). This strategy was based on an episomal plasmid that contained the CaCas9 gene, the sgRNA for the target gene and a resistance marker gene for selection. The aim of this project is the inactivation of the entire C. orthopsilosis ALS family using the plasmid-based CRISPR-Cas9 system, using a unique sgRNA to generate independent triple mutant strains in different genetic backgrounds. Firstly, as a proof of concept, the episomal CRISPR-Cas9 strategy (pSAT1_Ribo_CoADE2) was used to transform the sequenced isolate 90-125 to generate CoED-ade2/ade2 mutants, which formed pink colonies, using a repair template containing two stop codons and a recognition site for an endonuclease. Hence, edited colonies were visually screened for pink phenotypes and their genotype was confirmed by PCR, digestion and sequencing. Then a plasmid harbouring the CaCAS9 gene and a sgRNA able to simultaneously target a 20 nucleotides sequence shared among each member of the ALS gene family was constructed (pSAT1_Ribo_CoALS). The sequenced strain of C. orthopsilosis, 90-125, together with a clinical isolate, Co124, previously shown to be highly adherent to human buccal epithelial cells (HBECs), were selected as parental strains for transformation. pSAT1_Ribo_CoALS plasmid was then electroporated in C. orthopsilosis strains together with a repair template containing two stop codons as well as a unique restriction site, to quickly genotype the recombinant colonies. Therefore, CRISPR-edited clones lacking functional copies of the entire CoALS gene family were successfully obtained in two different C. orthopsilosis genetic backgrounds. Their genotype was confirmed by PCR, digestion and sequencing. Positive clones to all the screenings were named 90-125-3ED and Co124-3ED. Wild type and triple-mutant strains did not show any growing defect on liquid and solid media. CRISPR-edited clones were compared to their relative parental strain for their ability to adhere to HBECs. Notably, adhesion assay to HBECs revealed that the deletion of the entire ALS gene family had a striking effect on the adhesion ability of C. orthopsilosis to a biotic surface. In fact, when compared with their respective parental strain, both edited strains showed a significant reduction in the adhesion ability, with more than 80% and 90% reduction in the adhesion index, respectively. The results obtained demonstrate for the first time that the CRISPR-Cas9 system can be used for the efficient inactivation of an entire gene family with a one-step transformation in C. orthopsilosis, significantly speeding up the creation of multiple edited strains. Moreover, adhesion experiment indicated that the ALS gene family plays a pivotal role in the adhesion process of C. orthopsilosis to HBECs.