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
  • 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. [email protected].
  • 5. Department of Bioscience, TUM School of Natural Sciences and TUM School of Medicine,Technical University of Munich, Munich, Germany. [email protected].
  • # 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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