Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple <italic toggle="yes">Candida</italic> Species

ABSTRACT Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and t...

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Autores principales:	Lisa Lombardi, João Oliveira-Pacheco, Geraldine Butler
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	CRISPR Candida genome editing Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ee60b813f2a0425fbe47bc1702149b97
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spelling	oai:doaj.org-article:ee60b813f2a0425fbe47bc1702149b972021-11-15T15:22:21ZPlasmid-Based CRISPR-Cas9 Gene Editing in Multiple <italic toggle="yes">Candida</italic> Species10.1128/mSphere.00125-192379-5042https://doaj.org/article/ee60b813f2a0425fbe47bc1702149b972019-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00125-19https://doaj.org/toc/2379-5042ABSTRACT Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species. We describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis, and C. metapsilosis. We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis. We show that editing can easily be carried out in multiple C. tropicalis isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in C. metapsilosis and C. tropicalis. IMPORTANCE Candida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis. We have also constructed a separate system for gene editing in C. tropicalis.Lisa LombardiJoão Oliveira-PachecoGeraldine ButlerAmerican Society for MicrobiologyarticleCRISPRCandidagenome editingMicrobiologyQR1-502ENmSphere, Vol 4, Iss 2 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR Candida genome editing Microbiology QR1-502
spellingShingle	CRISPR Candida genome editing Microbiology QR1-502 Lisa Lombardi João Oliveira-Pacheco Geraldine Butler Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple <italic toggle="yes">Candida</italic> Species
description	ABSTRACT Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species. We describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis, and C. metapsilosis. We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis. We show that editing can easily be carried out in multiple C. tropicalis isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in C. metapsilosis and C. tropicalis. IMPORTANCE Candida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis. We have also constructed a separate system for gene editing in C. tropicalis.
format	article
author	Lisa Lombardi João Oliveira-Pacheco Geraldine Butler
author_facet	Lisa Lombardi João Oliveira-Pacheco Geraldine Butler
author_sort	Lisa Lombardi
title	Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple <italic toggle="yes">Candida</italic> Species
title_short	Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple <italic toggle="yes">Candida</italic> Species
title_full	Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple <italic toggle="yes">Candida</italic> Species
title_fullStr	Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple <italic toggle="yes">Candida</italic> Species
title_full_unstemmed	Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple <italic toggle="yes">Candida</italic> Species
title_sort	plasmid-based crispr-cas9 gene editing in multiple <italic toggle="yes">candida</italic> species
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/ee60b813f2a0425fbe47bc1702149b97
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Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple <italic toggle="yes">Candida</italic> Species

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