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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.

ABSTRACT: 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-?0, his3-?0, and lys9-?0 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.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC5844300 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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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.

Zhao Yu Y Boeke Jef D JD

G3 (Bethesda, Md.) 20180302 3

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 rec ...[more]

PMID: 29321167

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Project description:BackgroundConstruction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC), is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date.Methodology/principal findingsWe investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp). No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe.Conclusions/significanceWe concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different markers constructed in this research are available from NBRP-yeast (http://yeast.lab.nig.ac.jp/).

Dataset Information

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

Publications

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

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