Rapid and efficient CRISPR/Cas9-based mating-type switching of Saccharomyces cerevisiae

Ze Xiong Xie; Leslie A. Mitchell; Hui Min Liu; Bing Zhi Li; Duo Liu; Neta Agmon; Yi Wu; Xia Li; Xiao Zhou; Bo Li; Wen Hai Xiao; Ming Zhu Ding; Ying Wang; Ying Jin Yuan; Jef D. Boeke

doi:10.1534/g3.117.300347

Rapid and efficient CRISPR/Cas9-based mating-type switching of Saccharomyces cerevisiae

Ze Xiong Xie, Leslie A. Mitchell, Hui Min Liu, Bing Zhi Li, Duo Liu, Neta Agmon, Yi Wu, Xia Li, Xiao Zhou, Bo Li, Wen Hai Xiao, Ming Zhu Ding, Ying Wang, Ying Jin Yuan, Jef D. Boeke

Research output: Contribution to journal › Article › peer-review

Abstract

Rapid and highly efficient mating-type switching of Saccharomyces cerevisiae enables a wide variety of genetic manipulations, such as the construction of strains, for instance, isogenic haploid pairs of both mating-types, diploids and polyploids. We used the CRISPR/Cas9 system to generate a double-strand break at the MAT locus and, in a single cotransformation, both haploid and diploid cells were switched to the specified mating-type at ~80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking HMLa and HMRa cryptic mating loci. Furthermore, we transplanted the synthetic yeast chromosome V to build a haploid polysynthetic chromosome strain by using this method together with an endoreduplication intercross strategy. The CRISPR/Cas9 mating-type switching method will be useful in building the complete synthetic yeast (Sc2.0) genome. Importantly, it is a generally useful method to build polyploids of a defined genotype and generally expedites strain construction, for example, in the construction of fully a/a/α/α isogenic tetraploids.

Original language	English (US)
Pages (from-to)	173-183
Number of pages	11
Journal	G3: Genes, Genomes, Genetics
Volume	8
Issue number	1
DOIs	https://doi.org/10.1534/g3.117.300347
State	Published - Jan 1 2018

Keywords

CRISPR/Cas9
Mating-type switching
Polyploidy
Ring chromosome
Saccharomyces cerevisiae

ASJC Scopus subject areas

Molecular Biology
Genetics
Genetics(clinical)

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Rapid and efficient CRISPR/Cas9-based mating-type switching of Saccharomyces cerevisiae. / Xie, Ze Xiong; Mitchell, Leslie A.; Liu, Hui Min; Li, Bing Zhi; Liu, Duo; Agmon, Neta; Wu, Yi; Li, Xia; Zhou, Xiao; Li, Bo; Xiao, Wen Hai; Ding, Ming Zhu; Wang, Ying; Yuan, Ying Jin; Boeke, Jef D.

In: G3: Genes, Genomes, Genetics, Vol. 8, No. 1, 01.01.2018, p. 173-183.

Research output: Contribution to journal › Article › peer-review

Xie, Ze Xiong ; Mitchell, Leslie A. ; Liu, Hui Min ; Li, Bing Zhi ; Liu, Duo ; Agmon, Neta ; Wu, Yi ; Li, Xia ; Zhou, Xiao ; Li, Bo ; Xiao, Wen Hai ; Ding, Ming Zhu ; Wang, Ying ; Yuan, Ying Jin ; Boeke, Jef D. / Rapid and efficient CRISPR/Cas9-based mating-type switching of Saccharomyces cerevisiae. In: G3: Genes, Genomes, Genetics. 2018 ; Vol. 8, No. 1. pp. 173-183.

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title = "Rapid and efficient CRISPR/Cas9-based mating-type switching of Saccharomyces cerevisiae",

abstract = "Rapid and highly efficient mating-type switching of Saccharomyces cerevisiae enables a wide variety of genetic manipulations, such as the construction of strains, for instance, isogenic haploid pairs of both mating-types, diploids and polyploids. We used the CRISPR/Cas9 system to generate a double-strand break at the MAT locus and, in a single cotransformation, both haploid and diploid cells were switched to the specified mating-type at ~80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking HMLa and HMRa cryptic mating loci. Furthermore, we transplanted the synthetic yeast chromosome V to build a haploid polysynthetic chromosome strain by using this method together with an endoreduplication intercross strategy. The CRISPR/Cas9 mating-type switching method will be useful in building the complete synthetic yeast (Sc2.0) genome. Importantly, it is a generally useful method to build polyploids of a defined genotype and generally expedites strain construction, for example, in the construction of fully a/a/α/α isogenic tetraploids.",

keywords = "CRISPR/Cas9, Mating-type switching, Polyploidy, Ring chromosome, Saccharomyces cerevisiae",

author = "Xie, {Ze Xiong} and Mitchell, {Leslie A.} and Liu, {Hui Min} and Li, {Bing Zhi} and Duo Liu and Neta Agmon and Yi Wu and Xia Li and Xiao Zhou and Bo Li and Xiao, {Wen Hai} and Ding, {Ming Zhu} and Ying Wang and Yuan, {Ying Jin} and Boeke, {Jef D.}",

note = "Funding Information: to the Sc2.0 consortium. We dedicate this paper to the memory of Amar Klar. The work in China was supported by the Ministry of Science and Technology of China (grants 2014CB745100 and 2015DFA00960), and the National Natural Science Foundation of China (grants 21390203 and 21621004). The work in the United States was supported by National Science Foundation grants MCB-1026068 and MCB-1158201 to J.D.B. Funding Information: We thank Rodney Rothstein for the generous contribution of the set of centromere destabilizing constructs, originally developed in his laboratoryto the Sc2.0 consortium. We dedicate this paper to the memory of Amar Klar. The work in China was supported by the Ministry of Science and Technology of China (grants 2014CB745100 and 2015DFA00960), and the National Natural Science Foundation of China (grants 21390203 and 21621004). The work in the United States was supported by National Science Foundation grants MCB-1026068 and MCB-1158201 to J.D.B.",

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AU - Mitchell, Leslie A.

AU - Liu, Hui Min

AU - Li, Bing Zhi

AU - Liu, Duo

AU - Agmon, Neta

AU - Wu, Yi

AU - Li, Xia

AU - Zhou, Xiao

AU - Li, Bo

AU - Xiao, Wen Hai

AU - Ding, Ming Zhu

AU - Wang, Ying

AU - Yuan, Ying Jin

AU - Boeke, Jef D.

N1 - Funding Information: to the Sc2.0 consortium. We dedicate this paper to the memory of Amar Klar. The work in China was supported by the Ministry of Science and Technology of China (grants 2014CB745100 and 2015DFA00960), and the National Natural Science Foundation of China (grants 21390203 and 21621004). The work in the United States was supported by National Science Foundation grants MCB-1026068 and MCB-1158201 to J.D.B. Funding Information: We thank Rodney Rothstein for the generous contribution of the set of centromere destabilizing constructs, originally developed in his laboratoryto the Sc2.0 consortium. We dedicate this paper to the memory of Amar Klar. The work in China was supported by the Ministry of Science and Technology of China (grants 2014CB745100 and 2015DFA00960), and the National Natural Science Foundation of China (grants 21390203 and 21621004). The work in the United States was supported by National Science Foundation grants MCB-1026068 and MCB-1158201 to J.D.B.

PY - 2018/1/1

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N2 - Rapid and highly efficient mating-type switching of Saccharomyces cerevisiae enables a wide variety of genetic manipulations, such as the construction of strains, for instance, isogenic haploid pairs of both mating-types, diploids and polyploids. We used the CRISPR/Cas9 system to generate a double-strand break at the MAT locus and, in a single cotransformation, both haploid and diploid cells were switched to the specified mating-type at ~80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking HMLa and HMRa cryptic mating loci. Furthermore, we transplanted the synthetic yeast chromosome V to build a haploid polysynthetic chromosome strain by using this method together with an endoreduplication intercross strategy. The CRISPR/Cas9 mating-type switching method will be useful in building the complete synthetic yeast (Sc2.0) genome. Importantly, it is a generally useful method to build polyploids of a defined genotype and generally expedites strain construction, for example, in the construction of fully a/a/α/α isogenic tetraploids.

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