Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain

Abstract Kluyveromyces marxianus, a probiotic yeast, is important in industrial applications because it has a broad substrate spectrum, a rapid growth rate and high thermotolerance. To date, however, there has been little effort in its genetic engineering by the CRISPR/Cas9 system. Therefore, we aim...

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Autores principales:	Ming-Hsuan Lee, Jinn-Jy Lin, Yu-Ju Lin, Jui-Jen Chang, Huei-Mien Ke, Wen-Lang Fan, Tzi-Yuan Wang, Wen-Hsiung Li
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/6feaf2ff22e74f2eb8d7897cd6ad254d
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spelling	oai:doaj.org-article:6feaf2ff22e74f2eb8d7897cd6ad254d2021-12-02T11:40:17ZGenome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain10.1038/s41598-018-25366-z2045-2322https://doaj.org/article/6feaf2ff22e74f2eb8d7897cd6ad254d2018-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-25366-zhttps://doaj.org/toc/2045-2322Abstract Kluyveromyces marxianus, a probiotic yeast, is important in industrial applications because it has a broad substrate spectrum, a rapid growth rate and high thermotolerance. To date, however, there has been little effort in its genetic engineering by the CRISPR/Cas9 system. Therefore, we aimed at establishing the CRISPR/Cas9 system in K. marxianus and creating stable haploid strains, which will make genome engineering simpler. First, we predicted the genome-wide target sites of CRISPR/Cas9 that have been conserved among the eight sequenced genomes of K. marxianus strains. Second, we established the CRISPR/Cas9 system in the K. marxianus 4G5 strain, which was selected for its high thermotolerance, rapid growth, a pH range of pH3-9, utilization of xylose, cellobiose and glycerol, and toxin tolerance, and we knocked out its MATα3 to prevent mating-type switching. Finally, we used K. marxianus MATα3 knockout diploid strains to obtain stable haploid strains with a growth rate comparable to that of the diploid 4G5 strain. In summary, we present the workflow from identifying conserved CRISPR/Cas9 targets in the genome to knock out the MATα3 genes in K. marxianus to obtain a stable haploid strain, which can facilitate genome engineering applications.Ming-Hsuan LeeJinn-Jy LinYu-Ju LinJui-Jen ChangHuei-Mien KeWen-Lang FanTzi-Yuan WangWen-Hsiung LiNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-10 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ming-Hsuan Lee Jinn-Jy Lin Yu-Ju Lin Jui-Jen Chang Huei-Mien Ke Wen-Lang Fan Tzi-Yuan Wang Wen-Hsiung Li Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain
description	Abstract Kluyveromyces marxianus, a probiotic yeast, is important in industrial applications because it has a broad substrate spectrum, a rapid growth rate and high thermotolerance. To date, however, there has been little effort in its genetic engineering by the CRISPR/Cas9 system. Therefore, we aimed at establishing the CRISPR/Cas9 system in K. marxianus and creating stable haploid strains, which will make genome engineering simpler. First, we predicted the genome-wide target sites of CRISPR/Cas9 that have been conserved among the eight sequenced genomes of K. marxianus strains. Second, we established the CRISPR/Cas9 system in the K. marxianus 4G5 strain, which was selected for its high thermotolerance, rapid growth, a pH range of pH3-9, utilization of xylose, cellobiose and glycerol, and toxin tolerance, and we knocked out its MATα3 to prevent mating-type switching. Finally, we used K. marxianus MATα3 knockout diploid strains to obtain stable haploid strains with a growth rate comparable to that of the diploid 4G5 strain. In summary, we present the workflow from identifying conserved CRISPR/Cas9 targets in the genome to knock out the MATα3 genes in K. marxianus to obtain a stable haploid strain, which can facilitate genome engineering applications.
format	article
author	Ming-Hsuan Lee Jinn-Jy Lin Yu-Ju Lin Jui-Jen Chang Huei-Mien Ke Wen-Lang Fan Tzi-Yuan Wang Wen-Hsiung Li
author_facet	Ming-Hsuan Lee Jinn-Jy Lin Yu-Ju Lin Jui-Jen Chang Huei-Mien Ke Wen-Lang Fan Tzi-Yuan Wang Wen-Hsiung Li
author_sort	Ming-Hsuan Lee
title	Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain
title_short	Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain
title_full	Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain
title_fullStr	Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain
title_full_unstemmed	Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain
title_sort	genome-wide prediction of crispr/cas9 targets in kluyveromyces marxianus and its application to obtain a stable haploid strain
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/6feaf2ff22e74f2eb8d7897cd6ad254d
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Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain

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