RNA Inosine Sensor-Guided TadA Mutational Scanning for Toxicity Minimization of Adenine Base Editors.

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.