Optimized nickase- and nuclease-based prime editing in human and mouse cells

Fatwa Adikusuma, Caleb Lushington, Jayshen Arudkumar, Gelshan I. Godahewa, Yu C.J. Chey, Luke Gierus, Sandra Piltz, Ashleigh Geiger, Yatish Jain, Daniel Reti, Laurence O.W. Wilson, Denis C. Bauer, Paul Q. Thomas

Research output: Contribution to journalArticlepeer-review

41 Citations (Scopus)

Abstract

Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. In this study, we generated all-in-one prime editing (PEA1) constructs that carry all the components required for PE, along with a selection marker. We tested these constructs (with selection) in HEK293T, K562, HeLa and mouse embryonic stem (ES) cells. We discovered that PE efficiency in HEK293T cells was much higher than previously observed, reaching up to 95% (mean 67%). The efficiency in K562 and HeLa cells, however, remained low. To improve PE efficiency in K562 and HeLa, we generated a nuclease prime editor and tested this system in these cell lines as well as mouse ES cells. PE-nuclease greatly increased prime editing initiation, however, installation of the intended edits was often accompanied by extra insertions derived from the repair template. Finally, we show that zygotic injection of the nuclease prime editor can generate correct modifications in mouse fetuses with up to 100% efficiency.

Original languageEnglish
Pages (from-to)10785-10795
Number of pages11
JournalNucleic Acids Research
Volume49
Issue number18
DOIs
Publication statusPublished or Issued - 11 Oct 2021

ASJC Scopus subject areas

  • Genetics

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