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

Unlabelled: 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 . 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 replacing the , and 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, and . However, no improved editing or targeting efficiencies were detected in or backgrounds. Our research highlights important challenges in precise genome editing of 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 Summary: 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 infections. However, precise genetic manipulation in has proven difficult due to inefficient genome editing tools. This study compares four different CRISPR-based allele editing systems in , identifying their strengths and limitations. The findings provide crucial insights into selecting the best tools for genetic research in , guiding future efforts to combat this formidable pathogen.