A Cloning-Free Recyclable System for CRISPR-Cas9 Mediated Mutant and Reversion Construction in Candida albicans Clinical Isolates
Date of Award
Master of Science (MS)
Microbiology, Immunology, and Biochemistry
Brian Peters, PhD
Elizabeth A. Fitzpatrick, PhD; Jarrod R. Fortwendel, PhD; Camaron R. Hole, PhD
Candida, CRISPR, genetics, molecular genetics, molecular mycology, mycology
Candida albicans is a ubiquitous opportunistic fungal pathogen and one of the most prevalent causes of fungal diseases worldwide. The reference isolate SC5314 is one of the most widely used strains for both experimental and genetic studies, but it is becoming increasingly evident that genetic diversity in clinical isolates plays an important role in antifungal resistance, virulence, and pathogenicity. These recent discoveries highlight the need for genetic tools that are capable of investigating genes in multiple strain backgrounds. Here we build on the SAT1-flipper method and combine it with CRISPR-Cas9 technology to achieve cloning-free homozygous deletion in a single transformation event, followed by similarly efficient gene reversion. Short CRISPR RNAs (crRNAs) are combined with universal transactivating CRISPR RNA (tracrRNA) to form single guide RNA (sgRNA) that direct Cas9 to the target gene as part of the ribonucleoprotein (RNP) complex. To supplement the SAT1-flipper plasmid, we constructed an isogenic plasmid containing a C. albicans-optimized Hygromycin B resistance gene (CaHygB) in place of the SAT1 nourseothricin resistance gene to serve as repair templates. PCR-amplification of these cassettes with primers containing sequences homologous to the flanking regions of the target gene allow for high-efficiency homology-directed repair. We first targeted ADE2 for deletion, as homozygous deletion results in red pigmented colonies. In one transformation event, we achieved homozygous ADE2 deletion in SC5314 and a variety of clinical isolates (529L, JS15, SJCA1, and TW1) at a rate of nearly 100% when transformants were incubated on media containing both nourseothricin and hygromycin B. Gene reversion was similarly successful by engineering plasmids pDUP3 to contain the ADH1 terminator and an overlap extension PCR-mediated approach combined with CRISPR-Cas9 targeting at the NEUT5 locus. We also showed the efficacy of this method in deletion of the essential metabolic gene URA3 and the recyclability of the system by performing consecutive deletion of the morphology regulating genes CPH1 and EFG1. Thus, we have established an efficient, cloning-free, recyclable system for homozygous deletion and reversion across C. albicans isolates.
Vogel, Amanda K. (https://orcid.org/0000-0001-7002-1278), "A Cloning-Free Recyclable System for CRISPR-Cas9 Mediated Mutant and Reversion Construction in Candida albicans Clinical Isolates" (2022). Theses and Dissertations (ETD). Paper 625. http://dx.doi.org/10.21007/etd.cghs.2022.0612.