A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts
Methylotrophic yeasts such as <i>Ogataea polymorpha</i> and <i>Komagataella phaffii</i> (sin. <i>Hansenula polymorpha</i> and <i>Pichia pastoris</i>, respectively) are commonly used in basic research and biotechnological applications, frequently those...
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MDPI AG
2023-05-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/9/8173 |
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| author | Azamat V. Karginov Marina G. Tarutina Anastasia R. Lapteva Maria D. Pakhomova Artur A. Galliamov Sergey Y. Filkin Alexey N. Fedorov Michael O. Agaphonov |
| author_facet | Azamat V. Karginov Marina G. Tarutina Anastasia R. Lapteva Maria D. Pakhomova Artur A. Galliamov Sergey Y. Filkin Alexey N. Fedorov Michael O. Agaphonov |
| author_sort | Azamat V. Karginov |
| collection | DOAJ |
| description | Methylotrophic yeasts such as <i>Ogataea polymorpha</i> and <i>Komagataella phaffii</i> (sin. <i>Hansenula polymorpha</i> and <i>Pichia pastoris</i>, respectively) are commonly used in basic research and biotechnological applications, frequently those requiring genome modifications. However, the CRISPR-Cas9 genome editing approaches reported for these species so far are relatively complex and laborious. In this work we present an improved plasmid vector set for CRISPR-Cas9 genome editing in methylotrophic yeasts. This includes a plasmid encoding Cas9 with a nuclear localization signal and plasmids with a scaffold for the single guide RNA (sgRNA). Construction of a sgRNA gene for a particular target sequence requires only the insertion of a 24 bp oligonucleotide duplex into the scaffold. Prior to yeast transformation, each plasmid is cleaved at two sites, one of which is located within the selectable marker, so that the functional marker can be restored only via recombination of the Cas9-containing fragment with the sgRNA gene-containing fragment. This recombination leads to the formation of an autonomously replicating plasmid, which can be lost from yeast clones after acquisition of the required genome modification. The vector set allows the use of G418-resistance and <i>LEU2</i> auxotrophic selectable markers. The functionality of this setup has been demonstrated in <i>O. polymorpha</i>, <i>O. parapolymorpha</i>, <i>O. haglerorum</i> and <i>Komagataella phaffii</i>. |
| first_indexed | 2024-03-11T04:15:52Z |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T04:15:52Z |
| publishDate | 2023-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-1c0c9b011dbe4fb98486d71a9d6a88102023-11-17T23:05:55ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-05-01249817310.3390/ijms24098173A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic YeastsAzamat V. Karginov0Marina G. Tarutina1Anastasia R. Lapteva2Maria D. Pakhomova3Artur A. Galliamov4Sergey Y. Filkin5Alexey N. Fedorov6Michael O. Agaphonov7The Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences, Bach Institute of Biochemistry, 119071 Moscow, RussiaNational Research Center Kurchatov Institute, 123182 Moscow, RussiaNational Research Center Kurchatov Institute, 123182 Moscow, RussiaThe Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences, Bach Institute of Biochemistry, 119071 Moscow, RussiaThe Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences, Bach Institute of Biochemistry, 119071 Moscow, RussiaThe Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences, Bach Institute of Biochemistry, 119071 Moscow, RussiaThe Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences, Bach Institute of Biochemistry, 119071 Moscow, RussiaThe Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences, Bach Institute of Biochemistry, 119071 Moscow, RussiaMethylotrophic yeasts such as <i>Ogataea polymorpha</i> and <i>Komagataella phaffii</i> (sin. <i>Hansenula polymorpha</i> and <i>Pichia pastoris</i>, respectively) are commonly used in basic research and biotechnological applications, frequently those requiring genome modifications. However, the CRISPR-Cas9 genome editing approaches reported for these species so far are relatively complex and laborious. In this work we present an improved plasmid vector set for CRISPR-Cas9 genome editing in methylotrophic yeasts. This includes a plasmid encoding Cas9 with a nuclear localization signal and plasmids with a scaffold for the single guide RNA (sgRNA). Construction of a sgRNA gene for a particular target sequence requires only the insertion of a 24 bp oligonucleotide duplex into the scaffold. Prior to yeast transformation, each plasmid is cleaved at two sites, one of which is located within the selectable marker, so that the functional marker can be restored only via recombination of the Cas9-containing fragment with the sgRNA gene-containing fragment. This recombination leads to the formation of an autonomously replicating plasmid, which can be lost from yeast clones after acquisition of the required genome modification. The vector set allows the use of G418-resistance and <i>LEU2</i> auxotrophic selectable markers. The functionality of this setup has been demonstrated in <i>O. polymorpha</i>, <i>O. parapolymorpha</i>, <i>O. haglerorum</i> and <i>Komagataella phaffii</i>.https://www.mdpi.com/1422-0067/24/9/8173Cas9CRISPRgenome engineeringgenome editingmethylotrophic yeast<i>Ogataea</i> |
| spellingShingle | Azamat V. Karginov Marina G. Tarutina Anastasia R. Lapteva Maria D. Pakhomova Artur A. Galliamov Sergey Y. Filkin Alexey N. Fedorov Michael O. Agaphonov A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts International Journal of Molecular Sciences Cas9 CRISPR genome engineering genome editing methylotrophic yeast <i>Ogataea</i> |
| title | A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts |
| title_full | A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts |
| title_fullStr | A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts |
| title_full_unstemmed | A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts |
| title_short | A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts |
| title_sort | split marker system for crispr cas9 genome editing in methylotrophic yeasts |
| topic | Cas9 CRISPR genome engineering genome editing methylotrophic yeast <i>Ogataea</i> |
| url | https://www.mdpi.com/1422-0067/24/9/8173 |
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