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Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors

Vectors encoding selectable markers have been widely used in yeast to maintain or express exogenous DNA fragments. In the fission yeast Schizosaccharomyces pombe, several engineered markers have been reported and widely used, such as ura4(+) and ScLEU2 from Saccharomyces cerevisiae, which complement...

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Autores principales: Zhao, Yu, Boeke, Jef D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Genetics Society of America 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844300/
https://www.ncbi.nlm.nih.gov/pubmed/29321167
http://dx.doi.org/10.1534/g3.117.300363
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author Zhao, Yu
Boeke, Jef D.
author_facet Zhao, Yu
Boeke, Jef D.
author_sort Zhao, Yu
collection PubMed
description Vectors encoding selectable markers have been widely used in yeast to maintain or express exogenous DNA fragments. In the fission yeast Schizosaccharomyces pombe, several engineered markers have been reported and widely used, such as ura4(+) and ScLEU2 from Saccharomyces cerevisiae, which complement ura4 and leu1 mutations, respectively. These two auxotrophic markers share no homology with the S. pombe genome; however, most others can recombine with the genome due to sequence homology shared between the genomic and plasmid-borne copies of the markers. Here, we describe a CRISPR-Cas9 toolbox that can be used to quickly introduce “designer” auxotrophic marker deletions into host strains, including leu1-Δ0, his3-Δ0, and lys9-Δ0. Together with ura4-D18, this brings the total number of available designer deletion auxotrophic markers to four. The toolbox consists of a Cas9-gRNA expression vector and a donor DNA plasmid pair for each designer deletion. Using this toolbox, a set of auxotrophic S. pombe strains was constructed. Further, we reorganized essential components in the commonly used pREP series of plasmids and assembled the corresponding auxotrophic marker gene onto these plasmids. This toolbox for producing designer deletions, together with the newly developed strains and plasmids, will benefit the whole yeast community.
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spelling pubmed-58443002018-03-22 Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors Zhao, Yu Boeke, Jef D. G3 (Bethesda) Investigations Vectors encoding selectable markers have been widely used in yeast to maintain or express exogenous DNA fragments. In the fission yeast Schizosaccharomyces pombe, several engineered markers have been reported and widely used, such as ura4(+) and ScLEU2 from Saccharomyces cerevisiae, which complement ura4 and leu1 mutations, respectively. These two auxotrophic markers share no homology with the S. pombe genome; however, most others can recombine with the genome due to sequence homology shared between the genomic and plasmid-borne copies of the markers. Here, we describe a CRISPR-Cas9 toolbox that can be used to quickly introduce “designer” auxotrophic marker deletions into host strains, including leu1-Δ0, his3-Δ0, and lys9-Δ0. Together with ura4-D18, this brings the total number of available designer deletion auxotrophic markers to four. The toolbox consists of a Cas9-gRNA expression vector and a donor DNA plasmid pair for each designer deletion. Using this toolbox, a set of auxotrophic S. pombe strains was constructed. Further, we reorganized essential components in the commonly used pREP series of plasmids and assembled the corresponding auxotrophic marker gene onto these plasmids. This toolbox for producing designer deletions, together with the newly developed strains and plasmids, will benefit the whole yeast community. Genetics Society of America 2018-01-10 /pmc/articles/PMC5844300/ /pubmed/29321167 http://dx.doi.org/10.1534/g3.117.300363 Text en Copyright © 2018 Zhao, Boeke http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Investigations
Zhao, Yu
Boeke, Jef D.
Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors
title Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors
title_full Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors
title_fullStr Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors
title_full_unstemmed Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors
title_short Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors
title_sort construction of designer selectable marker deletions with a crispr-cas9 toolbox in schizosaccharomyces pombe and new design of common entry vectors
topic Investigations
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844300/
https://www.ncbi.nlm.nih.gov/pubmed/29321167
http://dx.doi.org/10.1534/g3.117.300363
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