Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Candida Species

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Many pathogenic Candida species possess diploid genomes and lack classical meiosis, complicating genetic manipulation.
• Existing clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems are not universally applicable across all Candida species.
• Efficient molecular tools are needed to study the genomes of medically significant Candida species like Candida parapsilosis and Candida tropicalis.

Purpose of the Study:

• To adapt and develop novel CRISPR-Cas9 gene editing systems for efficient genome manipulation in Candida species.
• To enable markerless gene editing, introduction of molecular barcodes, and generation of heterozygous mutations.
• To provide accessible gene editing tools for the Candida parapsilosis species group and Candida tropicalis.

Main Methods:

• Adaptation of a CRISPR-Cas9 system using an autonomously replicating plasmid for Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis.
• Development of a separate autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis.
• Utilized guide RNAs released by tRNA-ribozyme cleavage, co-expressed with CAS9 and a SAT1 marker.

Main Results:

• Successfully demonstrated markerless gene editing in Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis.
• Showcased the ability to introduce molecular barcodes and reintroduce wild-type sequences into edited strains.
• Developed and validated a distinct CRISPR-Cas9 system for efficient gene editing across multiple Candida tropicalis isolates, noting high levels of nonhomologous end joining (NHEJ) repair in C. metapsilosis and C. tropicalis.

Conclusions:

• The developed CRISPR-Cas9 systems provide simple, efficient, and versatile tools for genomic studies in medically important Candida species.
• These advancements facilitate the investigation of virulence factors and drug resistance mechanisms in Candida infections.
• The new systems address the need for advanced molecular manipulation methods in Candida research.