Dec 7, 2018

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Journal of Visualized Experiments : JoVE
Ben A EvansDouglas A Bernstein

Abstract

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans ...Continue Reading

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Mentioned in this Paper

Conserved Sequence
Candida castellii
Candida albicans
Cyclodictyon albicans
Genome
Daiotyla albicans
Genes
CRISPR-Cas Systems
Craspedia glabrata
Gene Editing

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