Genetic Transformation of Candida auris via Homology-Directed Repair Using a Standard Lithium Acetate Protocol

Gustavo Bravo Ruiz, Alexander Lorenz (Lead / Corresponding author)

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

Abstract

Reverse genetics is a particularly powerful tool in non-model organisms with known whole-genome sequences enabling the characterization of gene and, thus, protein function via a mutant phenotype. Reverse genetic approaches require genetic manipulation techniques which often need to be specifically developed for non-model organisms; this can be fraught with difficulties. Here, we describe a genetic transformation protocol for the recently emerged human pathogen Candida auris to target the integration of DNA constructs into genomic locations via homology-directed repair using long flanking homologous sequences (>1 kb). We detail the generation of DNA constructs for gene deletion with dominant drug resistance markers via fusion PCR, the transformation of these constructs into chemically competent C. auris cells, and the confirmation of correct integration by PCR. This strategy can be adapted to deliver DNA constructs other than deletion cassettes, including promoter exchanges and protein tags.

Original languageEnglish
Title of host publicationCandida auris
Subtitle of host publicationMethods and Protocols
EditorsAlexander Lorenz
PublisherSpringer
Chapter8
Pages95-110
Number of pages16
Edition1
ISBN (Electronic)9781071624173
ISBN (Print)9781071624166
DOIs
Publication statusPublished - 9 Jun 2022

Publication series

NameMethods in Molecular Biology
Volume2517
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Acetates
  • Antifungal Agents/metabolism
  • Candida/genetics
  • Candida auris
  • Polymerase Chain Reaction/methods
  • Transformation, Genetic
  • Gene targeting
  • Genetic transformation
  • Genetic manipulation
  • Dominant drug resistance markers
  • Gene deletion

Fingerprint

Dive into the research topics of 'Genetic Transformation of Candida auris via Homology-Directed Repair Using a Standard Lithium Acetate Protocol'. Together they form a unique fingerprint.

Cite this