A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 2; referees: 2 approved]
In the fission yeast Schizosaccharomyces pombe the prevailing approach for gene manipulations is based on homologous recombination of a PCR product that contains genomic target sequences and a selectable marker. The CRISPR/Cas9 system has recently been implemented in fission yeast, which allows for...
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| Format: | Article |
| Language: | English |
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Wellcome
2017-01-01
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| Series: | Wellcome Open Research |
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| Online Access: | https://wellcomeopenresearch.org/articles/1-19/v2 |
| _version_ | 1828416101004869632 |
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| author | María Rodríguez-López Cristina Cotobal Oscar Fernández-Sánchez Natalia Borbarán Bravo Risky Oktriani Heike Abendroth Dardan Uka Mimoza Hoti Jin Wang Mikel Zaratiegui Jürg Bähler |
| author_facet | María Rodríguez-López Cristina Cotobal Oscar Fernández-Sánchez Natalia Borbarán Bravo Risky Oktriani Heike Abendroth Dardan Uka Mimoza Hoti Jin Wang Mikel Zaratiegui Jürg Bähler |
| author_sort | María Rodríguez-López |
| collection | DOAJ |
| description | In the fission yeast Schizosaccharomyces pombe the prevailing approach for gene manipulations is based on homologous recombination of a PCR product that contains genomic target sequences and a selectable marker. The CRISPR/Cas9 system has recently been implemented in fission yeast, which allows for seamless genome editing without integration of a selection marker or leaving any other genomic ‘scars’. The published method involves manual design of the single guide RNA (sgRNA), and digestion of a large plasmid with a problematic restriction enzyme to clone the sgRNA. To increase the efficiency of this approach, we have established and optimized a PCR-based system to clone the sgRNA without restriction enzymes into a plasmid with a dominant natMX6 (nourseothricin) selection marker. We also provide a web-tool, CRISPR4P, to support the design of the sgRNAs and the primers required for the entire process of seamless DNA deletion. Moreover, we report the preparation of G1-synchronized and cryopreserved S. pombe cells, which greatly increases the efficiency and speed for transformations, and may also facilitate standard gene manipulations. Applying this optimized CRISPR/Cas9-based approach, we have successfully deleted over 80 different non-coding RNA genes, which are generally lowly expressed, and have inserted 7 point mutations in 4 different genomic regions. |
| first_indexed | 2024-12-10T13:57:02Z |
| format | Article |
| id | doaj.art-4606762220074b02ab82fcf96ef33dd7 |
| institution | Directory Open Access Journal |
| issn | 2398-502X |
| language | English |
| last_indexed | 2024-12-10T13:57:02Z |
| publishDate | 2017-01-01 |
| publisher | Wellcome |
| record_format | Article |
| series | Wellcome Open Research |
| spelling | doaj.art-4606762220074b02ab82fcf96ef33dd72022-12-22T01:45:55ZengWellcomeWellcome Open Research2398-502X2017-01-01110.12688/wellcomeopenres.10038.211382A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 2; referees: 2 approved]María Rodríguez-López0Cristina Cotobal1Oscar Fernández-Sánchez2Natalia Borbarán Bravo3Risky Oktriani4Heike Abendroth5Dardan Uka6Mimoza Hoti7Jin Wang8Mikel Zaratiegui9Jürg Bähler10Research Department of Genetics, Evolution and Environment, University College London, London, UKResearch Department of Genetics, Evolution and Environment, University College London, London, UKResearch Department of Genetics, Evolution and Environment, University College London, London, UKResearch Department of Genetics, Evolution and Environment, University College London, London, UKResearch Department of Genetics, Evolution and Environment, University College London, London, UKResearch Department of Genetics, Evolution and Environment, University College London, London, UKResearch Department of Genetics, Evolution and Environment, University College London, London, UKResearch Department of Genetics, Evolution and Environment, University College London, London, UKResearch Department of Genetics, Evolution and Environment, University College London, London, UKDepartment of Molecular Biology and Biochemistry, Rutgers University, Piscataway, USAResearch Department of Genetics, Evolution and Environment, University College London, London, UKIn the fission yeast Schizosaccharomyces pombe the prevailing approach for gene manipulations is based on homologous recombination of a PCR product that contains genomic target sequences and a selectable marker. The CRISPR/Cas9 system has recently been implemented in fission yeast, which allows for seamless genome editing without integration of a selection marker or leaving any other genomic ‘scars’. The published method involves manual design of the single guide RNA (sgRNA), and digestion of a large plasmid with a problematic restriction enzyme to clone the sgRNA. To increase the efficiency of this approach, we have established and optimized a PCR-based system to clone the sgRNA without restriction enzymes into a plasmid with a dominant natMX6 (nourseothricin) selection marker. We also provide a web-tool, CRISPR4P, to support the design of the sgRNAs and the primers required for the entire process of seamless DNA deletion. Moreover, we report the preparation of G1-synchronized and cryopreserved S. pombe cells, which greatly increases the efficiency and speed for transformations, and may also facilitate standard gene manipulations. Applying this optimized CRISPR/Cas9-based approach, we have successfully deleted over 80 different non-coding RNA genes, which are generally lowly expressed, and have inserted 7 point mutations in 4 different genomic regions.https://wellcomeopenresearch.org/articles/1-19/v2Bioinformatics |
| spellingShingle | María Rodríguez-López Cristina Cotobal Oscar Fernández-Sánchez Natalia Borbarán Bravo Risky Oktriani Heike Abendroth Dardan Uka Mimoza Hoti Jin Wang Mikel Zaratiegui Jürg Bähler A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 2; referees: 2 approved] Wellcome Open Research Bioinformatics |
| title | A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 2; referees: 2 approved] |
| title_full | A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 2; referees: 2 approved] |
| title_fullStr | A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 2; referees: 2 approved] |
| title_full_unstemmed | A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 2; referees: 2 approved] |
| title_short | A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 2; referees: 2 approved] |
| title_sort | crispr cas9 based method and primer design tool for seamless genome editing in fission yeast version 2 referees 2 approved |
| topic | Bioinformatics |
| url | https://wellcomeopenresearch.org/articles/1-19/v2 |
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