Improved prime editing allows for routine predictable gene editing in Physcomitrium patens
© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.
| Veröffentlicht in: | Journal of experimental botany. - 1985. - 74(2023), 19 vom: 13. Okt., Seite 6176-6187 |
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| 1. Verfasser: | |
| Weitere Verfasser: | , , , , , |
| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2023
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| Zugriff auf das übergeordnete Werk: | Journal of experimental botany |
| Schlagworte: | Journal Article Research Support, Non-U.S. Gov't Physcomitrium patens EpegRNA genome editing prime editing pseudoknot split prime editing RNA-Directed DNA Polymerase EC 2.7.7.49 |
| Zusammenfassung: | © The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. Efficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed prime editing approach, a modified CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] editing method, has reached the precision goal but its editing rate can be improved. We present an improved methodology that allows for routine prime editing in the model plant Physcomitrium patens, whilst exploring potential new prime editing improvements. Using a standardized protoplast transfection procedure, multiple prime editing guide RNA (pegRNA) structural and prime editor variants were evaluated targeting the APT reporter gene through direct plant selection. Together, enhancements of expression of the prime editor, modifications of the 3' extension of the pegRNA, and the addition of synonymous mutation in the reverse transcriptase template sequence of the pegRNA dramatically improve the editing rate without affecting the quality of the edits. Furthermore, we show that prime editing is amenable to edit a gene of interest through indirect selection, as demonstrated by the generation of a Ppdek10 mutant. Additionally, we determine that a plant retrotransposon reverse transcriptase enables prime editing. Finally, we show for the first time the possibility of performing prime editing with two independently coded peptides |
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| Beschreibung: | Date Completed 23.10.2023 Date Revised 24.10.2023 published: Print Citation Status MEDLINE |
| ISSN: | 1460-2431 |
| DOI: | 10.1093/jxb/erad189 |