Subotic, A. | bioRxiv Science

bioRxiv2025

A comparative evaluation of CRISPR-Cas9 allele editing systems in Candida auris: challenging research in a challenging bug

Candida auris is an emergent fungal pathogen of significant interest for molecular research because of its unique nosocomial persistence, high stress tolerance and common multidrug resistance. To investigate the molecular mechanisms of these or other phenotypes, 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. Alarmingly, 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 common random ectopic integration of the CRISPR cassette. Although the plasmid-based system showed a low transformation success compared to the other systems, it has the highest editing efficiency with 41.9% correct transformants on average. In an attempt to improve editing efficiencies of integration-based systems by silencing the non-homologous end joining (NHEJ) DNA repair pathway, we deleted two main NHEJ factors, KU70 and LIG4. However, no improved editing or targeting efficiencies were detected in ku7011, lig411, or ku7011/lig411 backgrounds. Our research highlights important challenges in precise genome editing of C. auris and sheds light on the advantages and limitations of several methods with the aim to guide scientists in selecting the most appropriate tool for molecular work in this enigmatic fungal pathogen. Author summaryCandida auris is a rapidly emerging fungal pathogen that poses serious challenges to global healthcare. Understanding the genetic mechanisms that underlie its nosocomial persistence, virulence, multidrug resistance and other traits is essential for developing new treatments and preventing the spread and burden of C. auris infections. However, precise genetic manipulation in C. auris has proven difficult due to inefficient genome editing tools. This study compares four different CRISPR-based allele editing systems in C. auris, identifying their strengths and limitations. The findings provide crucial insights into selecting the best tools for genetic research in C. auris, guiding future efforts to combat this formidable pathogen.

microbiology↗

bioRxiv2020

Genome-wide analysis of experimentally evolved Candida auris reveals multiple novel mechanisms of multidrug-resistance

Candida auris is globally recognized as an opportunistic fungal pathogen of high concern, due to its extensive multidrug-resistance (MDR). Still, molecular mechanisms of MDR are largely unexplored. This is the first account of genome wide evolution of MDR in C. auris obtained through serial in vitro exposure to azoles, polyenes and echinocandins. We show the stepwise accumulation of multiple novel mutations in genes known and unknown in antifungal drug resistance, albeit almost all new for C. auris. Echinocandin resistance evolved through a codon deletion in FKS1 accompanied by a substitution in FKS1 hot spot 3. Mutations in ERG3 and CIS2 further increased the echinocandin MIC. Decreased azole susceptibility was acquired through a gain of function mutation in transcription factor TAC1b yielding overexpression of the drug efflux pump Cdr1; a segmental duplication of chromosome 1 containing ERG11; and a whole chromosome 5 duplication, which contains TAC1b. The latter was associated with increased expression of ERG11, TAC1b and CDR2, but not CDR1. The simultaneous emergence of nonsense mutations in ERG3 and ERG11, presumably leading to the abrogation of ergosterol synthesis, was shown to decrease amphotericin B susceptibility, accompanied with fluconazole cross resistance. A mutation in MEC3, a gene mainly known for its role in DNA damage homeostasis, further increased the polyene MIC. Overall, this study shows the alarming potential and diversity for MDR development in C. auris, even in a clade until now not associated with MDR (clade II), hereby stressing its clinical importance and the urge for future research. ImportanceC. auris is a recently discovered human fungal pathogens and has shown an alarming potential for multi- and pan-resistance towards all classes of antifungals most commonly used in the clinic. Currently, C. auris has been globally recognized as a nosocomial pathogen of high concern due to this evolutionary potential. So far, this is the first study in which the stepwise progression of MDR in C. auris is monitored in vitro. Multiple novel mutations in known resistance genes and genes previously not or vaguely associated with drug resistance reveal rapid MDR evolution in a C. auris clade II isolate. Additionally, this study shows that in vitro experimental evolution can be a powerful tool to discover new drug resistance mechanisms, although it has its limitations.

microbiology↗