Harnessing nucleotide metabolism to control glycosylase base editing outcomes

TL;DR

This paper shows how adjusting nucleotide levels can improve the efficiency and accuracy of genome editing tools that change specific DNA bases.

Contribution

The study introduces a novel metabolic strategy using thymidine to enhance base editing outcomes in mammalian cells.

Findings

Elevating dTTP levels via thymidine supplementation increased C-to-A, G-to-A, and T-to-A editing efficiencies significantly.

dT treatment improved A-product purity and enabled efficient generation of pathogenic mutations in a disease model.

Supplementing with dA modestly increased T-to-A editing outcomes.

Abstract

Rationale: Glycosylase-derived base editors enable transversion base substitutions, expanding the scope of genome engineering for both basic research and clinical applications. However, the variable outcomes and low efficiency of B (C/G/T)-to-A editing in mammalian cells hinder their broader utility, likely due to inefficient thymine translesion synthesis (TLS) across apurinic/apyrimidinic (AP) sites.

Methods and Results: We developed a nucleotide metabolism-based strategy to enhance B-to-A editing by leveraging endogenous nucleotide metabolism. We showed that elevating intracellular deoxythymidine triphosphate (dTTP) levels via exogenous thymidine (dT) supplementation, which activates the thymidine kinase 1 (TK1)-dependent salvage pathway for the production of dTTP, increased C-to-A, G-to-A, and T-to-A editing efficiencies by up to 4-fold, 1.8-fold, and 1.8-fold, respectively, and improved A-product purity by up to 2.7-fold. Moreover, supplementation with dA increased T outcomes, albeit at a relatively modest level. In a disease-relevant single nucleotide variation (SNV) model, dT treatment enabled efficient generation of pathogenic mutations otherwise inaccessible to base editing.

Conclusion: Our findings establish metabolic modulation as a powerful means to control base editing outcomes and expand the functional capabilities of glycosylase-derived editors.

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