Format

Send to

Choose Destination
J Vis Exp. 2018 Nov 14;(141). doi: 10.3791/58764.

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

Author information

1
Department of Biology, Ball State University.
2
Whitehead Institute for Biomedical Research.
3
Department of Biology, Ball State University; dabernstein@bsu.edu.

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 CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

PMID:
30507925
DOI:
10.3791/58764

Supplemental Content

Full text links

Icon for MyJove Corporation
Loading ...
Support Center