2. Academic Validation
  3. Exonuclease-enhanced prime editors

Exonuclease-enhanced prime editors

  • Nat Methods. 2024 Feb 1. doi: 10.1038/s41592-023-02162-w.
Dong-Jiunn Jeffery Truong # 1 2,  Julian Geilenkeuser # 1 2,  Stephanie Victoria Wendel 1 2,  Julius Clemens Heinrich Wilming 1 2,  Niklas Armbrust 1 2,  Eva Maria Hildegard Binder 1 2,  Tobias Heinrich Santl 1 2,  Annika Siebenhaar 1 2,  Christoph Gruber 3,  Teeradon Phlairaharn 1 2,  Milica Živanić 1 2,  Gil Gregor Westmeyer 4 5
Affiliations

Affiliations

  • 1 Institute for Synthetic Biomedicine, Helmholtz Munich, Neuherberg, Germany.
  • 2 Department of Bioscience, TUM School of Natural Sciences and TUM School of Medicine,Technical University of Munich, Munich, Germany.
  • 3 Institute of Developmental Genetics, Helmholtz Munich, Neuherberg, Germany.
  • 4 Institute for Synthetic Biomedicine, Helmholtz Munich, Neuherberg, Germany. gil.westmeyer@tum.de.
  • 5 Department of Bioscience, TUM School of Natural Sciences and TUM School of Medicine,Technical University of Munich, Munich, Germany. gil.westmeyer@tum.de.
  • # Contributed equally.
PMID: 38302659 DOI: 10.1038/s41592-023-02162-w
Abstract

Prime editing (PE) is a powerful gene-editing technique based on targeted gRNA-templated reverse transcription and integration of the de novo synthesized single-stranded DNA. To circumvent one of the main bottlenecks of the method, the competition of the reverse-transcribed 3' FLAP with the original 5' FLAP DNA, we generated an enhanced fluorescence-activated cell sorting reporter cell line to develop an exonuclease-enhanced PE strategy ('Exo-PE') composed of an improved PE complex and an aptamer-recruited DNA-exonuclease to remove the 5' original DNA FLAP. Exo-PE achieved better overall editing efficacy than the reference PE2 strategy for insertions ≥30 base pairs in several endogenous loci and cell lines while maintaining the high editing precision of PE2. By enabling the precise incorporation of larger insertions, Exo-PE complements the growing palette of different PE tools and spurs additional refinements of the PE machinery.

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