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Truong, D.J.J. ; Geilenkeuser, J. ; Wendel, S.V. ; Wilming, J.C.H. ; Armbrust, N. ; Binder, E.M.H. ; Santl, T. ; Siebenhaar, A. ; Gruber, C. ; Phlairaharn, T. ; Živanić, M. ; Westmeyer, G.G.

Exonuclease-enhanced prime editors.

Nat. Methods 21, 455-464 (2024)
DOI PMC
Creative Commons Lizenzvertrag
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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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords Dna-binding; Protein; Bacteriophage-t5; Recognition; Mechanisms; Site; Rna
ISSN (print) / ISBN 1548-7091
e-ISSN 1548-7105
Journal Nature Methods
Quellenangaben Volume: 21, Issue: 3, Pages: 455-464 Article Number: , Supplement: ,
Publisher Nature Publishing Group
Publishing Place New York, NY
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Insitute of Synthetic Biomedicine (ISBM)
Institute of Developmental Genetics (IDG)
Grants
European Innovation Council (EIC Pathfinder)
European Research Council (ERC)
Free State of Bavaria under the Excellence Strategy of the Federal Government and the Laender through the ONE MUNICH project Munich Multiscale Biofabrication
Federal Ministry of Education and Research (BMBF)