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Acceleration of CRISPR/Cas9-Mediated Editing at Multiple Sites in the Saccharomyces cerevisiae Genome

The application of the CRISPR/Cas9-based genome editing technique to the yeast Saccharomyces cerevisiae has made it possible to simultaneously modify several sites, particularly to integrate several expression cassettes. The existing methods provide high efficiency for such modifications; however, c...

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Autores principales: Karpukhin, Alexey D., Sabirzyanov, Fanis A., Serebrianyi, Vsevolod A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10141985/
https://www.ncbi.nlm.nih.gov/pubmed/37104021
http://dx.doi.org/10.3390/mps6020039
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author Karpukhin, Alexey D.
Sabirzyanov, Fanis A.
Serebrianyi, Vsevolod A.
author_facet Karpukhin, Alexey D.
Sabirzyanov, Fanis A.
Serebrianyi, Vsevolod A.
author_sort Karpukhin, Alexey D.
collection PubMed
description The application of the CRISPR/Cas9-based genome editing technique to the yeast Saccharomyces cerevisiae has made it possible to simultaneously modify several sites, particularly to integrate several expression cassettes. The existing methods provide high efficiency for such modifications; however, common protocols include several preparatory steps, namely, the construction of an intermediate Cas9-expressing strain, the assembly of a plasmid bearing several single guide RNA (sgRNA) expression cassettes, and the surrounding integrated DNA fragments with long flanks for recombination with target loci. Since these preparatory steps are time consuming and may not be desirable in some types of experiments, we explored the possibility of multiple integration without these steps. We have demonstrated that it is possible to skip them simultaneously and integrate up to three expression cassettes into separate sites by transforming the recipient strain with the Cas9 expression plasmid, three differently marked sgRNA plasmids, and three donor DNAs flanked with short (70 bp) arms for recombination. This finding increases the flexibility of choosing the optimal experimental design for multiple editing of the genome of S. cerevisiae and can significantly accelerate such experiments.
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spelling pubmed-101419852023-04-29 Acceleration of CRISPR/Cas9-Mediated Editing at Multiple Sites in the Saccharomyces cerevisiae Genome Karpukhin, Alexey D. Sabirzyanov, Fanis A. Serebrianyi, Vsevolod A. Methods Protoc Article The application of the CRISPR/Cas9-based genome editing technique to the yeast Saccharomyces cerevisiae has made it possible to simultaneously modify several sites, particularly to integrate several expression cassettes. The existing methods provide high efficiency for such modifications; however, common protocols include several preparatory steps, namely, the construction of an intermediate Cas9-expressing strain, the assembly of a plasmid bearing several single guide RNA (sgRNA) expression cassettes, and the surrounding integrated DNA fragments with long flanks for recombination with target loci. Since these preparatory steps are time consuming and may not be desirable in some types of experiments, we explored the possibility of multiple integration without these steps. We have demonstrated that it is possible to skip them simultaneously and integrate up to three expression cassettes into separate sites by transforming the recipient strain with the Cas9 expression plasmid, three differently marked sgRNA plasmids, and three donor DNAs flanked with short (70 bp) arms for recombination. This finding increases the flexibility of choosing the optimal experimental design for multiple editing of the genome of S. cerevisiae and can significantly accelerate such experiments. MDPI 2023-04-04 /pmc/articles/PMC10141985/ /pubmed/37104021 http://dx.doi.org/10.3390/mps6020039 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Karpukhin, Alexey D.
Sabirzyanov, Fanis A.
Serebrianyi, Vsevolod A.
Acceleration of CRISPR/Cas9-Mediated Editing at Multiple Sites in the Saccharomyces cerevisiae Genome
title Acceleration of CRISPR/Cas9-Mediated Editing at Multiple Sites in the Saccharomyces cerevisiae Genome
title_full Acceleration of CRISPR/Cas9-Mediated Editing at Multiple Sites in the Saccharomyces cerevisiae Genome
title_fullStr Acceleration of CRISPR/Cas9-Mediated Editing at Multiple Sites in the Saccharomyces cerevisiae Genome
title_full_unstemmed Acceleration of CRISPR/Cas9-Mediated Editing at Multiple Sites in the Saccharomyces cerevisiae Genome
title_short Acceleration of CRISPR/Cas9-Mediated Editing at Multiple Sites in the Saccharomyces cerevisiae Genome
title_sort acceleration of crispr/cas9-mediated editing at multiple sites in the saccharomyces cerevisiae genome
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10141985/
https://www.ncbi.nlm.nih.gov/pubmed/37104021
http://dx.doi.org/10.3390/mps6020039
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