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Targeted Genetic Changes in Candida albicans Using Transient CRISPR‐Cas9 Expression
Author(s) -
Huang Manning Y.,
Cravener Max V.,
Mitchell Aaron P.
Publication year - 2021
Publication title -
current protocols
Language(s) - English
Resource type - Journals
ISSN - 2691-1299
DOI - 10.1002/cpz1.19
Subject(s) - crispr , biology , candida albicans , genotyping , computational biology , gene , crispr interference , virulence , genetics , genome editing , guide rna , transformation (genetics) , genotype
Candida albicans is an opportunistic fungal pathogen responsible for significant disease and mortality. Absent complete mating and other convenient methods, dissection of its virulence factors relies on robust tools to delete, complement, and otherwise modify genes of interest in this diploid organism. Here we describe the design principles and use of CRISPR associated nuclease 9 (Cas9) and single‐guide RNAs transiently expressed from PCR cassettes to modify genes of interest, generating homozygous mutants in a single transformation step. © 2021 Wiley Periodicals LLC. This article was corrected on 27 February 2023. See the end of the full text for details. Basic Protocol 1 : PCR amplification of CRISPR components Basic Protocol 2 : Transformation of Candida albicans Basic Protocol 3 : Selecting and genotyping transformants Alternate Protocol 1 : Deletion with recyclable markers by CRISPR induced marker excision (CRIME) Alternate Protocol 2 : Knock‐in and combining multiple cassettes with overlapping homology