In order to streamline the study of the pathogenic yeast Candida parapsilosis, we developed a plasmid-based CRISPR-Cas9 system (pRIBO) for gene editing in this species. However, pRIBO was not feasible for large-scale generation of mutant strains. We recently addressed this bottleneck by generating pCP-tRNA, an improved version of pRIBO in which the guide sequence can be easily cloned into the SapI-digested plasmid, and the release of the mature sgRNA is mediated by endogenous RNase cleavage of the C. parapsilosis tRNAAla, and by self-cleavage of the HDV ribozyme. We are currently using pCP-tRNA for the systematic generation of mutant strains. Suitable guides were computationally designed to induce Cas9 cleavage within the first 25 % of each ORF in the genome, which is then repaired by recombination with a repair template (RT) containing 30 bp homology arms, 11 bp to introduce a stop codon in the functional reading frame, and a unique tag. In 4 months, 288 plasmids targeting genes encoding transcription factors, phosphokinases, or unknown functions, were transformed into C. parapsilosis with the corresponding RTs. The system resulted in gene editing of 62 % of the 288 genes at high efficiency (80–100 % of the colonies tested were positive); 16 % of the genes in the panel may be essential based on homology with related species, and we believe that the remaining 22 % may be successfully edited by selecting a different guide. In conclusion, we demonstrate that pCP-tRNA is a valuable tool for high throughput generation of mutants in C. parapsilosis.

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