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CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans
Published on: November 14, 2018
PCR-based gene targeting in Candida albicans
Andrea Walther1, Jürgen Wendland
1Carlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10, DK-2500 Valby, Copenhagen, Denmark.
Nature Protocols
|September 6, 2008
Summary
This study introduces Rapid-PCR for faster gene targeting in Candida albicans. This method significantly reduces the time for creating gene deletion mutants, accelerating fungal research.
Area of Science:
- Mycology
- Molecular Biology
- Genetics
Background:
- PCR-based gene targeting accelerates gene function studies in fungi.
- Candida albicans is a diploid human fungal pathogen.
- Existing methods for gene targeting can be time-consuming.
Purpose of the Study:
- To describe a protocol utilizing Rapid-PCR for amplifying gene-targeting cassettes.
- To enable faster generation of gene deletion mutants in Candida albicans.
- To streamline the process of creating homozygous mutant strains.
Main Methods:
- Utilized Rapid-PCR to amplify pFA modules and cassettes.
- Optimized two alternative amplification conditions for cassette amplification.
- Employed Rapid-PCR on whole cells for rapid transformant identification, bypassing DNA extraction.
Main Results:
- Rapid-PCR amplified cassettes in 25-50 minutes, compared to 3-4 hours for standard PCR.
- Transformants were identified 2-4 days post-transformation.
- Complete allele deletion in C. albicans was achieved in under a week.
Conclusions:
- The Rapid-PCR protocol significantly accelerates gene targeting in Candida albicans.
- This method simplifies the generation of both heterozygous and homozygous mutants.
- The protocol enhances the efficiency of genetic manipulation in pathogenic fungi.
