In Candida auris, episomal editing worked better than genome-integration systems, which often failed to insert the editing cassette correctly.
Evidence
A systematic fungal genome-editing comparison across four CRISPR-Cas9 systems in C. auris found strain-dependent efficiencies, no correct linear cassette integration in more than 4,900 screened transformants for integration-based methods, and 41.9% correct transformants on average for the plasmid-based system.
Caveat
This is a methodological study in a fungal pathogen, and the main negative result was widespread ectopic rather than correct cassette integration, limiting confidence in integration-based editing workflows.
Simplified
Candidozyma (Candida) auris is an emergent fungal pathogen of significant interest for molecular research. A handful of based tools have been optimized for C. auris. Nonetheless, allele editing in this species remains a significant challenge, and different systems have different advantages and disadvantages. In this work, we compare four systems to introduce the genetic elements necessary for the production of Cas9 and the guide RNA molecule in the genome of C. auris, replacing the ENO1, LEU2 and HIS1 loci respectively, while the fourth system makes use of an . We observed that the editing efficiency of all four systems was significantly different and strain-dependent. However, we did not detect correct integration of linear CRISPR cassette constructs in integration-based systems, in over 4,900 screened transformants. Still, all transformants, whether correctly edited or not, grew on selective nourseothricin media, suggesting ectopic integration of the CRISPR cassette, which was confirmed by long-read whole genome sequencing. The plasmid-based system showed the highest editing efficiency with an average of 41.9% correct transformants, despite yielding fewer transformants compared to the other systems. Transformation of protoplasts or silencing the non-homologous end joining (NHEJ) DNA repair pathway, by deleting two main NHEJ factors, KU70 and LIG4, did not improve the editing efficiency. While our research highlights important challenges in precise genome editing of C. auris by quantitatively evaluating the editing and targeting efficiencies of different methods, it also clearly shows the safety and usefulness of plasmid-based systems like EPIC, which we recommend for molecular work in this enigmatic fungal pathogen.
Key numbers
41.9%
Correct Percentage
Average percentage of correct from the EPIC system.
6.5
per System
Average number of per transformation round for the EPIC system.
4,900
Count
Total number of screened for correct integration.
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