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Related Concept Videos

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CRISPR/Cas9 Genome Editing

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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Related Experiment Video

Updated: Jul 29, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Improved prime editing allows for routine predictable gene editing in Physcomitrium patens.

Pierre-François Perroud1, Anouchka Guyon-Debast1, Josep M Casacuberta2

  • 1Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB), 78000 Versailles, France.

Journal of Experimental Botany
|May 27, 2023
PubMed
Summary

Prime editing, a precise gene editing tool, has been enhanced for improved efficiency in the model plant Physcomitrium patens. This optimized method increases editing rates for genetic studies without compromising accuracy.

Keywords:
Physcomitrium patensEpegRNAgenome editingprime editingpseudoknotsplit prime editing

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Area of Science:

  • Molecular Biology
  • Plant Science
  • Genetics

Background:

  • Efficient and precise gene editing is crucial for reverse genetics.
  • Prime editing, a CRISPR/Cas9 derivative, offers precision but requires efficiency improvements.

Purpose of the Study:

  • To enhance the prime editing methodology for routine use in Physcomitrium patens.
  • To investigate strategies for improving prime editing efficiency and explore its applications.

Main Methods:

  • Standardized protoplast transfection in Physcomitrium patens.
  • Evaluation of various prime editing guide RNA (pegRNA) structures and prime editor variants.
  • Targeting the APT reporter gene for direct selection and Ppdek10 for indirect selection.

Main Results:

  • Optimized prime editor expression and pegRNA modifications significantly increased editing rates.
  • Synonymous mutations in the reverse transcriptase template enhanced efficiency without affecting edit quality.
  • Demonstrated successful gene editing via indirect selection and identified a plant retrotransposon reverse transcriptase for prime editing.
  • Showcased the potential for prime editing using two independently coded peptides.

Conclusions:

  • The developed methodology enables routine and efficient prime editing in Physcomitrium patens.
  • Enhancements in prime editor components and pegRNA design are key to improving editing rates.
  • Prime editing is a versatile tool applicable to both direct and indirect gene selection strategies in plants.