Efficient gene replacement by CRISPR/Cas-mediated homologous recombination in the model diatom Thalassiosira pseudonana
© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.
| Veröffentlicht in: | The New phytologist. - 1979. - 238(2023), 1 vom: 29. Apr., Seite 438-452 |
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| 1. Verfasser: | |
| Weitere Verfasser: | , , , , |
| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2023
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| Zugriff auf das übergeordnete Werk: | The New phytologist |
| Schlagworte: | Journal Article Research Support, Non-U.S. Gov't Thalassiosira pseudonana CRISPR algae diatom genome editing homology-directed repair recombination synthetic biology mehr... |
| Zusammenfassung: | © 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation. CRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life-cycle phase. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR in a diploid photosynthetic organism. Homologous recombination was induced in T. pseudonana using sequence-specific CRISPR/Cas, paired with a dsDNA donor matrix, generating substitution of the silacidin, nitrate reductase and urease genes by a resistance cassette (FCP:NAT). Up to c. 85% of NAT-resistant T. pseudonana colonies screened positive for HR by nested PCR. Precise integration of FCP:NAT at each locus was confirmed using an inverse PCR approach. The knockout of the nitrate reductase and urease genes impacted growth on nitrate and urea, respectively, while the knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell-size regulation in centric diatoms. Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and biotechnological applications targeted on harnessing the metabolic potential of diatoms |
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| Beschreibung: | Date Completed 06.03.2023 Date Revised 13.04.2023 published: Print-Electronic Citation Status MEDLINE |
| ISSN: | 1469-8137 |
| DOI: | 10.1111/nph.18587 |