A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9
Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within ge...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-10-01
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| Series: | Methods and Protocols |
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| Online Access: | https://www.mdpi.com/2409-9279/5/5/79 |
| _version_ | 1827648616858124288 |
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| author | Rhiannon R. Aguilar Zih-Jie Shen Jessica K. Tyler |
| author_facet | Rhiannon R. Aguilar Zih-Jie Shen Jessica K. Tyler |
| author_sort | Rhiannon R. Aguilar |
| collection | DOAJ |
| description | Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome. |
| first_indexed | 2024-03-09T19:41:40Z |
| format | Article |
| id | doaj.art-b5d54cbca0084d7688815a9c7810799a |
| institution | Directory Open Access Journal |
| issn | 2409-9279 |
| language | English |
| last_indexed | 2024-03-09T19:41:40Z |
| publishDate | 2022-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Methods and Protocols |
| spelling | doaj.art-b5d54cbca0084d7688815a9c7810799a2023-11-24T01:38:31ZengMDPI AGMethods and Protocols2409-92792022-10-01557910.3390/mps5050079A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9Rhiannon R. Aguilar0Zih-Jie Shen1Jessica K. Tyler2Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY 10065, USADepartment of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY 10065, USADepartment of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY 10065, USAUntil recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome.https://www.mdpi.com/2409-9279/5/5/79CRISPR-Cas9budding yeasthomologous recombinationpoint mutationgenome editinggenetic engineering |
| spellingShingle | Rhiannon R. Aguilar Zih-Jie Shen Jessica K. Tyler A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 Methods and Protocols CRISPR-Cas9 budding yeast homologous recombination point mutation genome editing genetic engineering |
| title | A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 |
| title_full | A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 |
| title_fullStr | A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 |
| title_full_unstemmed | A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 |
| title_short | A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 |
| title_sort | simple improved method for scarless genome editing of budding yeast using crispr cas9 |
| topic | CRISPR-Cas9 budding yeast homologous recombination point mutation genome editing genetic engineering |
| url | https://www.mdpi.com/2409-9279/5/5/79 |
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