Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in <named-content content-type="genus-species">Candida albicans</named-content>

ABSTRACT Most of our knowledge relating to molecular mechanisms of human fungal pathogenesis in Candida albicans relies on reverse genetics approaches, requiring strain engineering. DNA-mediated transformation of C. albicans has been described as highly mutagenic, potentially accentuated by the orga...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Timea Marton, Corinne Maufrais, Christophe d’Enfert, Melanie Legrand
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	CRISPR-Cas9 Candida albicans genome rearrangements Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/cdc40270ef2740f2a5459b3e3b97d217
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:cdc40270ef2740f2a5459b3e3b97d217
record_format	dspace
spelling	oai:doaj.org-article:cdc40270ef2740f2a5459b3e3b97d2172021-11-15T15:30:59ZUse of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in <named-content content-type="genus-species">Candida albicans</named-content>10.1128/mSphere.00620-202379-5042https://doaj.org/article/cdc40270ef2740f2a5459b3e3b97d2172020-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00620-20https://doaj.org/toc/2379-5042ABSTRACT Most of our knowledge relating to molecular mechanisms of human fungal pathogenesis in Candida albicans relies on reverse genetics approaches, requiring strain engineering. DNA-mediated transformation of C. albicans has been described as highly mutagenic, potentially accentuated by the organism’s genome plasticity, including the acquisition of genomic rearrangements, notably upon exposure to stress. The advent of CRISPR-Cas9 has vastly accelerated the process of genetically modifying strains, especially in diploid (such as C. albicans) and polyploid organisms. The effects of unleashing this nuclease within the genome of C. albicans are unknown, although several studies in other organisms report Cas9-associated toxicity and off-target DNA breaks. Upon the construction of a C. albicans strain collection, we took the opportunity to compare strains which were constructed using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation strategies, by quantifying and describing transformation-induced loss-of-heterozygosity and hyperploidy events. Our analysis of 57 strains highlights the mutagenic effects of transformation in C. albicans, regardless of the transformation protocol, but also underscores interesting differences in terms of genomic changes between strains obtained using different transformation protocols. Indeed, although strains constructed using the CRISPR-Cas9-free transformation method display numerous concomitant genomic changes randomly distributed throughout their genomes, the use of CRISPR-Cas9 leads to a reduced overall number of genome changes, particularly hyperploidies. Overall, in addition to facilitating strain construction by reducing the number of transformation steps, the CRISPR-Cas9-dependent transformation strategy in C. albicans appears to limit transformation-associated genome changes. IMPORTANCE Genome editing is essential to nearly all research studies aimed at gaining insight into the molecular mechanisms underlying various biological processes, including those in the opportunistic pathogen Candida albicans. The adaptation of the CRISPR-Cas9 system greatly facilitates genome engineering in many organisms. However, our understanding of the effects of CRISPR-Cas9 technology on the biology of C. albicans is limited. In this study, we sought to compare the extents of transformation-induced genomic changes within strains engineered using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation methods. CRISPR-Cas9-dependent transformation allows one to simultaneously target both homologs and, importantly, appears less mutagenic in C. albicans, since strains engineered using CRISPR-Cas9 display an overall decrease in concomitant genomic changes.Timea MartonCorinne MaufraisChristophe d’EnfertMelanie LegrandAmerican Society for MicrobiologyarticleCRISPR-Cas9Candida albicansgenome rearrangementsMicrobiologyQR1-502ENmSphere, Vol 5, Iss 5 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR-Cas9 Candida albicans genome rearrangements Microbiology QR1-502
spellingShingle	CRISPR-Cas9 Candida albicans genome rearrangements Microbiology QR1-502 Timea Marton Corinne Maufrais Christophe d’Enfert Melanie Legrand Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in <named-content content-type="genus-species">Candida albicans</named-content>
description	ABSTRACT Most of our knowledge relating to molecular mechanisms of human fungal pathogenesis in Candida albicans relies on reverse genetics approaches, requiring strain engineering. DNA-mediated transformation of C. albicans has been described as highly mutagenic, potentially accentuated by the organism’s genome plasticity, including the acquisition of genomic rearrangements, notably upon exposure to stress. The advent of CRISPR-Cas9 has vastly accelerated the process of genetically modifying strains, especially in diploid (such as C. albicans) and polyploid organisms. The effects of unleashing this nuclease within the genome of C. albicans are unknown, although several studies in other organisms report Cas9-associated toxicity and off-target DNA breaks. Upon the construction of a C. albicans strain collection, we took the opportunity to compare strains which were constructed using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation strategies, by quantifying and describing transformation-induced loss-of-heterozygosity and hyperploidy events. Our analysis of 57 strains highlights the mutagenic effects of transformation in C. albicans, regardless of the transformation protocol, but also underscores interesting differences in terms of genomic changes between strains obtained using different transformation protocols. Indeed, although strains constructed using the CRISPR-Cas9-free transformation method display numerous concomitant genomic changes randomly distributed throughout their genomes, the use of CRISPR-Cas9 leads to a reduced overall number of genome changes, particularly hyperploidies. Overall, in addition to facilitating strain construction by reducing the number of transformation steps, the CRISPR-Cas9-dependent transformation strategy in C. albicans appears to limit transformation-associated genome changes. IMPORTANCE Genome editing is essential to nearly all research studies aimed at gaining insight into the molecular mechanisms underlying various biological processes, including those in the opportunistic pathogen Candida albicans. The adaptation of the CRISPR-Cas9 system greatly facilitates genome engineering in many organisms. However, our understanding of the effects of CRISPR-Cas9 technology on the biology of C. albicans is limited. In this study, we sought to compare the extents of transformation-induced genomic changes within strains engineered using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation methods. CRISPR-Cas9-dependent transformation allows one to simultaneously target both homologs and, importantly, appears less mutagenic in C. albicans, since strains engineered using CRISPR-Cas9 display an overall decrease in concomitant genomic changes.
format	article
author	Timea Marton Corinne Maufrais Christophe d’Enfert Melanie Legrand
author_facet	Timea Marton Corinne Maufrais Christophe d’Enfert Melanie Legrand
author_sort	Timea Marton
title	Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in <named-content content-type="genus-species">Candida albicans</named-content>
title_short	Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in <named-content content-type="genus-species">Candida albicans</named-content>
title_full	Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in <named-content content-type="genus-species">Candida albicans</named-content>
title_fullStr	Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in <named-content content-type="genus-species">Candida albicans</named-content>
title_full_unstemmed	Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in <named-content content-type="genus-species">Candida albicans</named-content>
title_sort	use of crispr-cas9 to target homologous recombination limits transformation-induced genomic changes in <named-content content-type="genus-species">candida albicans</named-content>
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/cdc40270ef2740f2a5459b3e3b97d217
work_keys_str_mv	AT timeamarton useofcrisprcas9totargethomologousrecombinationlimitstransformationinducedgenomicchangesinnamedcontentcontenttypegenusspeciescandidaalbicansnamedcontent AT corinnemaufrais useofcrisprcas9totargethomologousrecombinationlimitstransformationinducedgenomicchangesinnamedcontentcontenttypegenusspeciescandidaalbicansnamedcontent AT christophedenfert useofcrisprcas9totargethomologousrecombinationlimitstransformationinducedgenomicchangesinnamedcontentcontenttypegenusspeciescandidaalbicansnamedcontent AT melanielegrand useofcrisprcas9totargethomologousrecombinationlimitstransformationinducedgenomicchangesinnamedcontentcontenttypegenusspeciescandidaalbicansnamedcontent
_version_	1718427854652309504

Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in <named-content content-type="genus-species">Candida albicans</named-content>

Ejemplares similares