Targeted Genetic Changes in Candida albicans Using Transient CRISPR‐Cas9 Expression

Abstract

Candida albicans is an opportunistic fungal pathogen responsible for significant disease and mortality. Absent complete mating and other convenient methods, dissection of its virulence factors relies on robust tools to delete, complement, and otherwise modify genes of interest in this diploid organism. Here we describe the design principles and use of CRISPR associated nuclease 9 (Cas9) and single-guide RNAs transiently expressed from PCR cassettes to modify genes of interest, generating homozygous mutants in a single transformation step.

The glycerophosphocholine acyltransferase Gpc1 contributes to phosphatidylcholine biosynthesis, long-term viability, and embedded hyphal growth in Candida albicans

“…Positive clones were selected for on LB agar containing 100 μg/ml ampicillin and confirmed through digestions with HindIII and Xho1. The final plasmid was then digested with NcoI prior to transformation into gpc1 Δ/Δ where it integrates at the RPS10 locus ( 60 ). Positive clones were selected on YNB lacking uracil.…”

CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms

“…Transcription factor mutants, tac1 ∆∆ and mrr2 ∆∆, were obtained from the Homann deletion collection provided by the Fungal Genetics Stock Center ( 17 ). The cdr1 ∆∆, tac1 ∆∆ mrr2 ∆ and tac1 ∆∆ mrr2 ∆∆ cdr1 ∆∆ mutants (see Table S1 for strains) were generated using transient CRISPR methods described by Huang et al ( 18 ). The mutants were confirmed using PCR analysis of the marker junctions and lacked the targeted ORF by PCR analysis; see Table S2 for primers used for these analyses.…”

Genetic Screening of Candida albicans Inactivation Mutants Identifies New Genes Involved in Macrophage-Fungal Cell Interactions

“…A second step for extension of the chimeric primers was run in order to fuse both PCR products ( Vyas et al, 2015 ). Finally, a nested PCR amplification with an outer upstream SRN52 forward primer and a downstream reverse primer from the ENO1 terminator, generated a nearly 1425 bp sgRNA cassette ( Huang et al, 2021 ).…”