Increasing intracellular dNTP levels improves prime editing efficiency

Pengpeng Liu; Karthikeyan Ponnienselvan; Thomas Nyalile; Sarah Oikemus; Anya T Joynt; Sukanya Iyer; Karen Kelly; Dongsheng Guo; Pyae P Kyawe; Emma Vanderleeden; Sambra D Redick; Lei Huang; Zexiang Chen; Jeong Min Lee; Celia A Schiffer; David M Harlan; Jennifer P Wang; Charles P Emerson Jr; Nathan D Lawson; Jonathan K Watts; Erik J Sontheimer; Jeremy Luban; Scot A Wolfe

doi:10.1038/s41587-024-02405-x

Increasing intracellular dNTP levels improves prime editing efficiency

Nat Biotechnol. 2025 Apr;43(4):539-544. doi: 10.1038/s41587-024-02405-x. Epub 2024 Sep 25.

Authors

Pengpeng Liu^#¹, Karthikeyan Ponnienselvan^#¹, Thomas Nyalile², Sarah Oikemus¹, Anya T Joynt¹, Sukanya Iyer¹, Karen Kelly³, Dongsheng Guo⁴, Pyae P Kyawe^{5

6}, Emma Vanderleeden^{5

6}, Sambra D Redick^{2

6}, Lei Huang¹, Zexiang Chen³, Jeong Min Lee⁷, Celia A Schiffer⁷, David M Harlan^{5

6}, Jennifer P Wang^{5

6}, Charles P Emerson Jr⁴, Nathan D Lawson^{1

2

8}, Jonathan K Watts^{3

7

8}, Erik J Sontheimer^{2

3

8}, Jeremy Luban^{2

3

7

8}, Scot A Wolfe^{9

10

11}

Affiliations

¹ Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
² Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA.
³ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA.
⁴ Department of Neurology, Wellstone Program, University of Massachusetts Chan Medical School, Worcester, MA, USA.
⁵ Department of Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA.
⁶ Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA.
⁷ Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
⁸ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA.
⁹ Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA. scot.wolfe@umassmed.edu.
¹⁰ Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA, USA. scot.wolfe@umassmed.edu.
¹¹ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA. scot.wolfe@umassmed.edu.

^# Contributed equally.

Abstract

In primary cell types, intracellular deoxynucleotide triphosphate (dNTP) levels are tightly regulated in a cell cycle-dependent manner. We report that prime editing efficiency is increased by mutations that improve the enzymatic properties of Moloney murine leukemia virus reverse transcriptase and treatments that increase intracellular dNTP levels. In combination, these modifications produce substantial increases in precise editing rates.

MeSH terms

Animals
Deoxyribonucleotides* / metabolism
Gene Editing* / methods
Humans
Mice
Moloney murine leukemia virus / enzymology
Moloney murine leukemia virus / genetics
Mutation
RNA-Directed DNA Polymerase* / genetics
RNA-Directed DNA Polymerase* / metabolism

Substances

Deoxyribonucleotides
RNA-Directed DNA Polymerase

Abstract

MeSH terms

Substances

Grants and funding