Improved prime editing allows for routine predictable gene editing in Physcomitrium patens
Perroud, Pierre-François (Université Paris-Saclay)
Guyon-Debast, Anouchka (Université Paris-Saclay)
Casacuberta i Suñer, Josep M 1962- (Centre de Recerca en Agrigenòmica)
Paul, Wyatt (Centre de Recherche de Chappes)
Pichon, Jean-Philippe (Centre de Recherche de Chappes)
Comeau, David (Centre de Recherche de Chappes)
Nogué, Fabien (Université Paris-Saclay)

Data:	2023
Resum:	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 3ʹ 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.
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	EpegRNA ; Genome editing ; Physcomitrium patens ; Prime editing ; Pseudoknot ; Split prime editing
Publicat a:	Journal of experimental botany, Vol. 74, Issue 19 (October 2023) , p. 6176-6187, ISSN 1460-2431

DOI: 10.1093/jxb/erad189
PMID: 37243510

12 p, 2.0 MB

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