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<p><i>Candidozyma</i> (<i>Candida</i>) <i>auris</i> is an emergent fungal pathogen of significant interest for molecular research. A handful of CRISPR-Cas9 based allele editing tools have been optimized for <i>C. auris</i>. 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 <i>C. auris</i>, replacing the <i>ENO1</i>, <i>LEU2</i> and <i>HIS1</i> loci respectively, while the fourth system makes use of an episomal plasmid. 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, <i>KU70</i> and <i>LIG4</i>, did not improve the editing efficiency. While our research highlights important challenges in precise genome editing of <i>C. auris</i> by quantitatively evaluating the editing and targeting efficiencies of different methods, it also clearly shows the safety and usefulness of plasmid-based systems like <i>EPIC</i>, which we recommend for molecular work in this enigmatic fungal pathogen.</p>

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A systematic comparison of CRISPR-Cas9 allele editing in Candida auris demonstrates unreliable cassette integration and effective episomal plasmid-based editing

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月8日

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  • Dimitrios Sofras,
  • Hans Carolus,
  • Ana Subotić,
  • Celia Lobo Romero,
  • Craig L. Ennis,
  • Lize Cuypers,
  • Katrien Lagrou,
  • Aaron D. Hernday,
  • Clarissa J. Nobile,
  • Jeffrey M. Rybak,
  • Patrick Van Dijck

摘要

Candidozyma (Candida) auris is an emergent fungal pathogen of significant interest for molecular research. A handful of CRISPR-Cas9 based allele editing 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 episomal plasmid. 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.