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Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role...

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Autores principales: Gervais, Nicholas C, La Bella, Alyssa A, Wensing, Lauren F, Sharma, Jehoshua, Acquaviva, Victoria, Best, Madison, Cadena López, Ricardo Omar, Fogal, Meea, Uthayakumar, Deeva, Chavez, Alejandro, Santiago-Tirado, Felipe, Flores-Mireles, Ana L, Shapiro, Rebecca S
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9911074/
https://www.ncbi.nlm.nih.gov/pubmed/36450451
http://dx.doi.org/10.1093/g3journal/jkac301
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author Gervais, Nicholas C
La Bella, Alyssa A
Wensing, Lauren F
Sharma, Jehoshua
Acquaviva, Victoria
Best, Madison
Cadena López, Ricardo Omar
Fogal, Meea
Uthayakumar, Deeva
Chavez, Alejandro
Santiago-Tirado, Felipe
Flores-Mireles, Ana L
Shapiro, Rebecca S
author_facet Gervais, Nicholas C
La Bella, Alyssa A
Wensing, Lauren F
Sharma, Jehoshua
Acquaviva, Victoria
Best, Madison
Cadena López, Ricardo Omar
Fogal, Meea
Uthayakumar, Deeva
Chavez, Alejandro
Santiago-Tirado, Felipe
Flores-Mireles, Ana L
Shapiro, Rebecca S
author_sort Gervais, Nicholas C
collection PubMed
description For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPR activation to overexpress genes involved in pathogenesis and drug susceptibility, and contribute toward the identification of novel phenotypes. Consequently, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.
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spelling pubmed-99110742023-02-13 Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans Gervais, Nicholas C La Bella, Alyssa A Wensing, Lauren F Sharma, Jehoshua Acquaviva, Victoria Best, Madison Cadena López, Ricardo Omar Fogal, Meea Uthayakumar, Deeva Chavez, Alejandro Santiago-Tirado, Felipe Flores-Mireles, Ana L Shapiro, Rebecca S G3 (Bethesda) Fungal Genetics and Genomics For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPR activation to overexpress genes involved in pathogenesis and drug susceptibility, and contribute toward the identification of novel phenotypes. Consequently, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression. Oxford University Press 2022-12-01 /pmc/articles/PMC9911074/ /pubmed/36450451 http://dx.doi.org/10.1093/g3journal/jkac301 Text en © The Author(s) 2022. Published by Oxford University Press on behalf of the Genetics Society of America. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Fungal Genetics and Genomics
Gervais, Nicholas C
La Bella, Alyssa A
Wensing, Lauren F
Sharma, Jehoshua
Acquaviva, Victoria
Best, Madison
Cadena López, Ricardo Omar
Fogal, Meea
Uthayakumar, Deeva
Chavez, Alejandro
Santiago-Tirado, Felipe
Flores-Mireles, Ana L
Shapiro, Rebecca S
Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans
title Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans
title_full Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans
title_fullStr Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans
title_full_unstemmed Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans
title_short Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans
title_sort development and applications of a crispr activation system for facile genetic overexpression in candida albicans
topic Fungal Genetics and Genomics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9911074/
https://www.ncbi.nlm.nih.gov/pubmed/36450451
http://dx.doi.org/10.1093/g3journal/jkac301
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