Cargando…

New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans

Precise genome modification is essential for the molecular dissection of Candida albicans, and is yielding invaluable information about the roles of specific gene functions in this major fungal pathogen of humans. C. albicans is naturally diploid, unable to undergo meiosis, and utilizes a non-canoni...

Descripción completa

Detalles Bibliográficos
Autores principales: Shahana, Shahida, Childers, Delma S., Ballou, Elizabeth R., Bohovych, Iryna, Odds, Frank C., Gow, Neil A. R., Brown, Alistair J. P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4062495/
https://www.ncbi.nlm.nih.gov/pubmed/24940603
http://dx.doi.org/10.1371/journal.pone.0100390
_version_ 1782321661448552448
author Shahana, Shahida
Childers, Delma S.
Ballou, Elizabeth R.
Bohovych, Iryna
Odds, Frank C.
Gow, Neil A. R.
Brown, Alistair J. P.
author_facet Shahana, Shahida
Childers, Delma S.
Ballou, Elizabeth R.
Bohovych, Iryna
Odds, Frank C.
Gow, Neil A. R.
Brown, Alistair J. P.
author_sort Shahana, Shahida
collection PubMed
description Precise genome modification is essential for the molecular dissection of Candida albicans, and is yielding invaluable information about the roles of specific gene functions in this major fungal pathogen of humans. C. albicans is naturally diploid, unable to undergo meiosis, and utilizes a non-canonical genetic code. Hence, specialized tools have had to be developed for gene disruption in C. albicans that permit the deletion of both target alleles, and in some cases, the recycling of the Candida-specific selectable markers. Previously, we developed a tool based on the Cre recombinase, which recycles markers in C. albicans with 90–100% efficiency via site-specific recombination between loxP sites. Ironically, the utility of this system was hampered by the extreme efficiency of Cre, which prevented the construction in Escherichia coli of stable disruption cassettes carrying a methionine-regulatable CaMET3(p)-cre gene flanked by loxP sites. Therefore, we have significantly enhanced this system by engineering new Clox cassettes that carry a synthetic, intron-containing cre gene. The Clox kit facilitates efficient transformation and marker recycling, thereby simplifying and accelerating the process of gene disruption in C. albicans. Indeed, homozygous mutants can be generated and their markers resolved within two weeks. The Clox kit facilitates strategies involving single marker recycling or multi-marker gene disruption. Furthermore, it includes the dominant NAT1 marker, as well as URA3, HIS1 and ARG4 cassettes, thereby permitting the manipulation of clinical isolates as well as genetically marked strains of C. albicans. The accelerated gene disruption strategies afforded by this new Clox system are likely to have a profound impact on the speed with which C. albicans pathobiology can be dissected.
format Online
Article
Text
id pubmed-4062495
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-40624952014-06-24 New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans Shahana, Shahida Childers, Delma S. Ballou, Elizabeth R. Bohovych, Iryna Odds, Frank C. Gow, Neil A. R. Brown, Alistair J. P. PLoS One Research Article Precise genome modification is essential for the molecular dissection of Candida albicans, and is yielding invaluable information about the roles of specific gene functions in this major fungal pathogen of humans. C. albicans is naturally diploid, unable to undergo meiosis, and utilizes a non-canonical genetic code. Hence, specialized tools have had to be developed for gene disruption in C. albicans that permit the deletion of both target alleles, and in some cases, the recycling of the Candida-specific selectable markers. Previously, we developed a tool based on the Cre recombinase, which recycles markers in C. albicans with 90–100% efficiency via site-specific recombination between loxP sites. Ironically, the utility of this system was hampered by the extreme efficiency of Cre, which prevented the construction in Escherichia coli of stable disruption cassettes carrying a methionine-regulatable CaMET3(p)-cre gene flanked by loxP sites. Therefore, we have significantly enhanced this system by engineering new Clox cassettes that carry a synthetic, intron-containing cre gene. The Clox kit facilitates efficient transformation and marker recycling, thereby simplifying and accelerating the process of gene disruption in C. albicans. Indeed, homozygous mutants can be generated and their markers resolved within two weeks. The Clox kit facilitates strategies involving single marker recycling or multi-marker gene disruption. Furthermore, it includes the dominant NAT1 marker, as well as URA3, HIS1 and ARG4 cassettes, thereby permitting the manipulation of clinical isolates as well as genetically marked strains of C. albicans. The accelerated gene disruption strategies afforded by this new Clox system are likely to have a profound impact on the speed with which C. albicans pathobiology can be dissected. Public Library of Science 2014-06-18 /pmc/articles/PMC4062495/ /pubmed/24940603 http://dx.doi.org/10.1371/journal.pone.0100390 Text en © 2014 Shahana et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Shahana, Shahida
Childers, Delma S.
Ballou, Elizabeth R.
Bohovych, Iryna
Odds, Frank C.
Gow, Neil A. R.
Brown, Alistair J. P.
New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
title New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
title_full New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
title_fullStr New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
title_full_unstemmed New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
title_short New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans
title_sort new clox systems for rapid and efficient gene disruption in candida albicans
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4062495/
https://www.ncbi.nlm.nih.gov/pubmed/24940603
http://dx.doi.org/10.1371/journal.pone.0100390
work_keys_str_mv AT shahanashahida newcloxsystemsforrapidandefficientgenedisruptionincandidaalbicans
AT childersdelmas newcloxsystemsforrapidandefficientgenedisruptionincandidaalbicans
AT ballouelizabethr newcloxsystemsforrapidandefficientgenedisruptionincandidaalbicans
AT bohovychiryna newcloxsystemsforrapidandefficientgenedisruptionincandidaalbicans
AT oddsfrankc newcloxsystemsforrapidandefficientgenedisruptionincandidaalbicans
AT gowneilar newcloxsystemsforrapidandefficientgenedisruptionincandidaalbicans
AT brownalistairjp newcloxsystemsforrapidandefficientgenedisruptionincandidaalbicans