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[Preprint]. 2023 Oct 29:2023.10.21.563443.
doi: 10.1101/2023.10.21.563443.

Addressing the dNTP bottleneck restricting prime editing activity

Karthikeyan Ponnienselvan, Pengpeng Liu, Thomas Nyalile, Sarah Oikemus, Anya T Joynt, Karen Kelly, Dongsheng Guo, Zexiang Chen, Jeong Min Lee, Celia A Schiffer, Charles P Emerson, Nathan D Lawson, Jonathan K Watts, Erik J Sontheimer, Jeremy Luban, Scot A Wolfe

Addressing the dNTP bottleneck restricting prime editing activity

Karthikeyan Ponnienselvan et al. bioRxiv. .

Update in

  • Increasing intracellular dNTP levels improves prime editing efficiency.
    Liu P, Ponnienselvan K, Nyalile T, Oikemus S, Joynt AT, Iyer S, Kelly K, Guo D, Kyawe PP, Vanderleeden E, Redick SD, Huang L, Chen Z, Lee JM, Schiffer CA, Harlan DM, Wang JP, Emerson CP Jr, Lawson ND, Watts JK, Sontheimer EJ, Luban J, Wolfe SA. Liu P, et al. Nat Biotechnol. 2025 Apr;43(4):539-544. doi: 10.1038/s41587-024-02405-x. Epub 2024 Sep 25. Nat Biotechnol. 2025. PMID: 39322763 Free PMC article.

Abstract

Prime editing efficiency is modest in cells that are quiescent or slowly proliferating where intracellular dNTP levels are tightly regulated. MMLV-reverse transcriptase - the prime editor polymerase subunit - requires high intracellular dNTPs levels for efficient polymerization. We report that prime editing efficiency in primary cells and in vivo is increased by mutations that enhance the enzymatic properties of MMLV-reverse transcriptase and can be further complemented by targeting SAMHD1 for degradation.

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