Rapid Hypothesis Testing in Candida albicans Clinical Isolates Using a Cloning-Free, Modular, and Recyclable System for CRISPR-Cas9 Mediated Mutant and Revertant Construction

Abstract

Recently, phenotypic heterogeneity in Candida albicans isolates has been recognized as an underappreciated factor contributing to gene diversification and broadly impacts strain-to-strain antifungal resistance, fitness, and pathogenicity. We have designed a cloning-free genetic system for rapid gene deletion and reversion in C. albicans clinical isolates that interlaces established recyclable genetic systems with CRISPR-Cas9 technology.

Intense pulsed light for inactivating planktonic and biofilm molds in food

“…Another strategy of the molds to resist extreme environments is the formation of biofilms. Biofilms generally consisting of exopolysaccharides, proteins, and nucleic acids, can strongly adheres to abiotic or biotic surfaces ( Lin et al, 2017 ; Liu et al, 2022a , c ). Biofilms are more resistant to antibiotics and biocidal agents ( Xu et al, 2011 ; Miao et al, 2019 ; Li et al, 2020a ).…”

A fungal metabolic regulator underlies infectious synergism duringCandida albicans-Staphylococcusaureus intra-abdominal co-infection

“…To determine whether RBK1 (a putative downstream target of Zcf13p) impacted agr activation during co-culture, rbk1 Δ/Δ and rbk1 Δ/Δ+ RBK1 strains were constructed as described ( Supplementary Methods ) 41, 42 . Similar to zcf13 Δ/Δ, the rbk1 Δ/Δ mutant led to reduced agr activation during co-culture and this was reverted to WT-levels with the rbk1 Δ/Δ+ RBK1 strain ( Fig.…”

Glycogen Metabolism in Candida albicans Impacts Fitness and Virulence during Vulvovaginal and Invasive Candidiasis

“…As we repeatedly could not generate a gdb1Δ/Δ strain by means of the standard techniques described above, we utilized CRISPR-Cas9 gene editing to create this strain and its revertant Δ/Δgdb1+GDB1 . Similarly, we constructed the double-knockout strain gph1Δ/Δ sga1Δ/Δ (from the gph1Δ/Δ strain) and deleted SGA1 or GPH1 from single revertants to generate gph1Δ/Δ sga1Δ/Δ+GPH1 and gph1Δ/Δ sga1Δ/Δ+SGA1 , using CRISPR-Cas9 as described previously ( 63 ). In brief, the disruption repair templates were amplified with the primers GOI_CC9KO-F+GOI_CC9KO-R, using SAT1 -flipper and CaHygB -flipper plasmids, which harbored resistance to nourseothricin and hygromycin, respectively.…”

“…After selection under a lower antibiotic pressure (YPD agar plates containing 25 μg/mL nourseothricin and 75 μg/mL of hygromycin), the correct genomic integration and the excision of the resistance cassettes were confirmed via the growth phenotype on YPD, YPD + 200 μg/mL nourseothricin, and YPD + 600 μg/mL hygromycin plates as well as via PCR amplification using the primers FRT_F+GOI_AmpR, FRT_R+GOI_AmpF, and GOI_DETF+GOI_DETR. In order to generate the gdb1Δ/Δ + GDB1 revertant, a repair template was PCR amplified by overlap extension PCR, which fused part of the NEUT5 promoter and ORF region of GDB1 and the ADH1 terminator, which were amplified from either the GP-1 gDNA or the pDUP3-tAHD1 plasmid, as described previously ( 63 ). The Δ/Δ gdb1 strain was edited with the CRISPR-Cas9 protocol, as described above, except that crNEUT5pDUPup crRNA was used during the RNP assembly.…”