Nature communicationsMay 9, 2025

Blocking DNA-PK and Polθ improves accuracy of different prime editing methods

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Jul 2, 2026

Abstract

Pharmacological inhibition of and Polϵ improves the precision of various systems.

Prime editing can produce unintended editing outcomes, including indels and imprecise edits.
Systems that enhance prime editing efficiency, like PE3 and PE5, often compromise precision.
Inhibiting DNA-PK and Polϵ can enhance the precision of prime editing across multiple genomic targets.
This approach reduces both unrelated indels and prime editing by-products, such as template duplications.
For the PEn system, co-inhibition also significantly improves the off-target editing profile.
Small molecule modulation of DNA repair pathways could be a general strategy to enhance prime editing outcomes.

Simplified

is a genome engineering tool that allows installation of various small edits with high precision. However, prime editing efficiency and purity can vary widely across different edits, genomic targets, and cell types. Prime editing typically offers more precise editing outcomes compared to other genome editing methods such as homology-directed repair. However, it can still result in significant rates of unintended editing outcomes, such as indels or imprecise prime edits. This issue is particularly notable in systems utilizing a second nicking gRNA, such as PE3 and PE5, as well as in dual pegRNA systems and fully active nuclease systems such as PEn, which increase efficiency but compromise precision. In this work, we show that pharmacological inhibition of and Polϴ, two major mediators of mutagenic DNA repair pathways, improves precision of PEn, PE3, PE5, PE7, and dual pegRNA editing systems, including TwinPE, HOPE, and Bi-PE, across multiple genomic loci and edit types. We show that co-inhibition of DNA-PK and Polϴ mitigates both prime editing-unrelated indels and prime editing by-products such as template duplications. Moreover, in the case of PEn, this strategy also substantially improved the off-target editing profile. Together, our data establish small molecule modulation of DNA repair pathways as a general strategy to maximize the precision of diverse prime editing systems.

Key numbers

93.4%

Precision Increase

Precision of PE3 with dual inhibitors in HEK293T cells.

48.6% to 91.9%

Indel Reduction

Indel rates in HeLa cells with 2iPE treatment.

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Blocking DNA-PK and Polθ improves accuracy of different prime editing methods

Abstract

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