Improved prime editing allows for routine predictable gene editing in Physcomitrium patens
Open accessAbstract
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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Prime editing provides significant improvements in genome editing by enabling precise DNA modifications-including targeted insertions, deletions, and substitutions-without causing double-strand breaks in plants.
“Prime editing (PE) provides significant improvements in genome editing by enabling precise DNA modifications—including targeted insertions, deletions, and substitutions—without causing double-strand breaks in plants (Kim et al., 2023; Perroud et al., 2023; Xu Y. et al., 2023).”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Perroud et al., 2023
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- Authors
- Perroud, P.-F., Guyon-Debast, A., Casacuberta, J. M., Paul, W., Pichon, J.-P., Comeau, D., & Nogué, F.
- Affiliations
- AgroParisTech (FR)
- Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB) , 78000 Versailles , France
- Universitat Autònoma de Barcelona (ES)
- Limagrain (France) (FR)
- Corresponding author
- Perroud, Pierre-François
- Venue
- Journal of Experimental Botany
- Publisher
- Oxford University Press (OUP)
- Published
- 2023-05-27
- Volume
- 74
- Issue
- 19
- Pages
- 6176-6187
- ISSN
- 0022-0957 (ISSN-L)
- 1460-2431
- PMC
- PMC10575697
- OpenAlex
- W4378533386
- Cited by
- 22
- References
- 47
- Citations by year
- 2026: 6 · 2025: 9 · 2024: 5 · 2023: 2
- FWCI
- 1.95
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Experimental Botany (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- HAL (submittedVersion, cc-by-nc)
- HAL (submittedVersion, cc-by)
- Dipòsit Digital de Documents de la UAB (Universitat Autònoma de Barcelona) (submittedVersion, cc-by)
- DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion, cc-by)
- Funders
- Limagrain Europe
- CRISPLUS
- The Institut Jean-Pierre Bourgin
- LabEx Saclay Plant Sciences
- Agence Nationale de la Recherche ((ANR)-10-LABX-0040-SPS)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Genetic and Mutation Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- prime editing, CRISPR/Cas9, Physcomitrella patens, bryophytes, genome editing efficiency, pegRNA design, protoplast transfection, reverse genetics
- MeSH terms
- Gene Editing, RNA-Directed DNA Polymerase, CRISPR-Cas Systems
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