Exonuclease-enhanced prime editors

Dong Jiunn Jeffery Truong, Julian Geilenkeuser, Stephanie Victoria Wendel, Julius Clemens Heinrich Wilming, Niklas Armbrust, Eva Maria Hildegard Binder, Tobias Heinrich Santl, Annika Siebenhaar, Christoph Gruber, Teeradon Phlairaharn, Milica Živanić, Gil Gregor Westmeyer

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

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.

Original languageEnglish
Pages (from-to)455-464
Number of pages10
JournalNature Methods
Volume21
Issue number3
DOIs
StatePublished - Mar 2024
Externally publishedYes

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