A ( ) -optimized Episomal Plasmid Induced Cas9-editing system reveals the direct impact of the S639F encoding mutation.

OBJECTIVES

Mutations in the ( ) β-glucan synthase gene ( ) altering S639 are frequently associated with clinical echinocandin resistance. However, the direct impact of these mutations remains uncharacterized. We have developed a novel optimized pisomal lasmid- nduced as9 (EPIC) gene-editing system capable of recyclable precision genome editing and demonstrate the contribution of mutation to echinocandin resistance.

METHODS

The EPIC gene-editing system was generated for optimized use in , and modification was evaluated in 5 clades. Mutations leading to Fks1 and Fks1 were placed into echinocandin-susceptible and echinocandin-resistant isolates from Clade-III and -I, respectively, using the EPIC system. Echinocandin susceptibility was determined by CLSI broth microdilution, and cell wall abundance of chitin and β-glucan was assessed by staining with Calcofluor White (CFW) and Aniline Blue (AB).

RESULTS

The EPIC system was capable of targeted editing in isolates from 5 genetic clades and shown to be precise by confirmatory sequencing of 50 transformants. A single nucleotide change in resulting in either the S639F substitution or a silent synonymous mutation was introduced in an echinocandin-susceptible Clade-III isolate. Precision editing by the EPIC system was confirmed by whole genome sequencing. Subsequent susceptibility testing demonstrated introduction of the S639F mutation to increase resistance to echinocandins. Moreover, introduction of a wildtype Fks1 sequence to an echinocandin-resistant Clade-I clinical isolate, correcting the mutation, resulted in a restoration of echinocandin sensitivity. Evaluation of cell wall composition showed isolates or strains harboring to contain significantly elevated β-glucan and chitin content relative to isogenic comparators.

CONCLUSIONS

These data demonstrate the potential of our EPIC system in its ability to introduce single nucleotide substitutions in multiple clade backgrounds while revealing the direct impact of the S639F encoding mutation on echinocandin resistance.