Molecular therapy : the journal of the American Society of Gene TherapyOct 6, 2025

Using RNA sensors to guide mutations in adenine base editors for reducing toxicity

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 13, 2026

Abstract

RNA off-target editing of classical adenine base editors (ABEs) is associated with substantial toxicity in vitro and in vivo.

The extent of RNA editing-related toxicity in ABEs remains unclear.
A novel fluorescent RNA inosine sensor was developed to evaluate RNA off-target editing efficiently.
Deep mutation scanning identified various TadA8e mutants with reduced RNA editing activity.
The H52L/D53R mutant is compatible with both SpCas9 and IscB nickase.
Engineered ABEs demonstrated the ability to target clinically relevant sites with improved precision.

Simplified

The TadA component of adenine base editors (ABEs) induces widespread RNA off-target edits and raises safety concerns for their applications. However, the extent of RNA editing-related toxicity remains elusive, and high-throughput engineering of ABEs focusing on RNA editing activities remains challenging. Here, we demonstrate that RNA off-target editing of classical ABEs leads to substantial toxicity in vitro and in vivo. We then design a rapid, cost-effective, and sensitive fluorescent RNA inosine sensor to accelerate RNA off-target editing evaluation and high-throughput screening in mammalian cells. Deep mutation scanning with the RNA sensor identifies various TadA8e mutants displaying minimized RNA editing activity, with the representative H52L/D53R mutant compatible with both SpCas9 and the compact IscB nickase. We show that the engineered ABEs could efficiently target clinically relevant sites in vitro and in vivo with enhanced precision, thereby providing promising tools for applications in which RNA editing-related toxicity should be carefully evaluated and minimized.

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Competing interests

Declaration of interests T.-L.C. and C. Zhang declare competing interests. Patent applications (No. 2025106198902, No. 2024113800475) on this work have been filed.

PubMed

Using RNA sensors to guide mutations in adenine base editors for reducing toxicity

Abstract

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Abstract

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