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CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus
BACKGROUND: The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces euba...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5718131/ https://www.ncbi.nlm.nih.gov/pubmed/29207996 http://dx.doi.org/10.1186/s12934-017-0835-1 |
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author | de Vries, Arthur R. Gorter de Groot, Philip A. van den Broek, Marcel Daran, Jean-Marc G. |
author_facet | de Vries, Arthur R. Gorter de Groot, Philip A. van den Broek, Marcel Daran, Jean-Marc G. |
author_sort | de Vries, Arthur R. Gorter |
collection | PubMed |
description | BACKGROUND: The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S. cerevisiae. RESULTS: This study reports a new CRISPR-Cas9 method for accurate gene deletion in S. pastorianus. This method combined the Streptococcus pyogenes cas9 gene expressed from either a chromosomal locus or from a mobile genetic element in combination with a plasmid-borne gRNA expression cassette. While the well-established gRNA expression system using the RNA polymerase III dependent SNR52 promoter failed, expression of a gRNA flanked with Hammerhead and Hepatitis Delta Virus ribozymes using the RNA polymerase II dependent TDH3 promoter successfully led to accurate deletion of all four alleles of the SeILV6 gene in strain CBS1483. Furthermore the expression of two ribozyme-flanked gRNAs separated by a 10-bp linker in a polycistronic array successfully led to the simultaneous deletion of SeATF1 and SeATF2, genes located on two separate chromosomes. The expression of this array resulted in the precise deletion of all five and four alleles mediated by homologous recombination in the strains CBS1483 and Weihenstephan 34/70 respectively, demonstrating the multiplexing abilities of this gRNA expression design. CONCLUSIONS: These results firmly established that CRISPR-Cas9 significantly facilitates and accelerates genome editing in S. pastorianus. |
format | Online Article Text |
id | pubmed-5718131 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-57181312017-12-08 CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus de Vries, Arthur R. Gorter de Groot, Philip A. van den Broek, Marcel Daran, Jean-Marc G. Microb Cell Fact Research BACKGROUND: The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S. cerevisiae. RESULTS: This study reports a new CRISPR-Cas9 method for accurate gene deletion in S. pastorianus. This method combined the Streptococcus pyogenes cas9 gene expressed from either a chromosomal locus or from a mobile genetic element in combination with a plasmid-borne gRNA expression cassette. While the well-established gRNA expression system using the RNA polymerase III dependent SNR52 promoter failed, expression of a gRNA flanked with Hammerhead and Hepatitis Delta Virus ribozymes using the RNA polymerase II dependent TDH3 promoter successfully led to accurate deletion of all four alleles of the SeILV6 gene in strain CBS1483. Furthermore the expression of two ribozyme-flanked gRNAs separated by a 10-bp linker in a polycistronic array successfully led to the simultaneous deletion of SeATF1 and SeATF2, genes located on two separate chromosomes. The expression of this array resulted in the precise deletion of all five and four alleles mediated by homologous recombination in the strains CBS1483 and Weihenstephan 34/70 respectively, demonstrating the multiplexing abilities of this gRNA expression design. CONCLUSIONS: These results firmly established that CRISPR-Cas9 significantly facilitates and accelerates genome editing in S. pastorianus. BioMed Central 2017-12-05 /pmc/articles/PMC5718131/ /pubmed/29207996 http://dx.doi.org/10.1186/s12934-017-0835-1 Text en © The Author(s) 2017 Open AccessThis article is 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 you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research de Vries, Arthur R. Gorter de Groot, Philip A. van den Broek, Marcel Daran, Jean-Marc G. CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus |
title | CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus |
title_full | CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus |
title_fullStr | CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus |
title_full_unstemmed | CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus |
title_short | CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus |
title_sort | crispr-cas9 mediated gene deletions in lager yeast saccharomyces pastorianus |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5718131/ https://www.ncbi.nlm.nih.gov/pubmed/29207996 http://dx.doi.org/10.1186/s12934-017-0835-1 |
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