Yuan Kexin, Xi Xin, Han Shaoqing, Han Jingyu, Zhao Bin, Wei Qi, Zhou Xiang
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, Hubei 430072, PR China.
State Key Laboratory of Metabolism and Regulation in Complex Organisms, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, Hubei 430071, PR China.
Nucleic Acids Res. 2025 Apr 10;53(7). doi: 10.1093/nar/gkaf281.
Adenosine base editors (ABEs) facilitate A·T to G·C base pair conversion with significant therapeutic potential for correcting pathogenic point mutations in human genetic diseases, such as sickle cell anemia and β-thalassemia. Unlike CRISPR-Cas9 systems that induce double-strand breaks, ABEs operate through precise deamination, avoiding chromosomal instability. However, the off-target editing effects of ABEs remain inadequately characterized. In this study, we present a biochemical method Selict-seq, designed to evaluate genome-wide off-target editing by ABEs. Selict-seq specifically captures deoxyinosine-containing single-stranded DNA and precisely identifies deoxyadenosine-to-deoxyinosine (dA-to-dI) mutation sites, elucidating the off-target effects induced by ABEs. Through investigations involving three single-guide RNAs, we identified numerous unexpected off-target edits both within and outside the protospacer regions. Notably, ABE8e(V106W) exhibited distinct off-target characteristics, including high editing rates (>10%) at previously unreported sites (e.g. RNF2 and EMX1) and out-of-protospacer mutations. These findings significantly advance our understanding of the off-target landscape associated with ABEs. In summary, our approach enables an unbiased analysis of the ABE editome and provides a widely applicable tool for specificity evaluation of various emerging genome editing technologies that produce intermediate products as deoxyinosine.
腺苷碱基编辑器（ABEs）可促进A·T到G·C碱基对的转换，在纠正人类遗传疾病（如镰状细胞贫血和β地中海贫血）的致病点突变方面具有巨大的治疗潜力。与诱导双链断裂的CRISPR-Cas9系统不同，ABEs通过精确脱氨发挥作用，避免了染色体不稳定。然而，ABEs的脱靶编辑效应仍未得到充分表征。在本研究中，我们提出了一种生化方法Selict-seq，旨在评估ABEs在全基因组范围内的脱靶编辑。Selict-seq专门捕获含脱氧次黄嘌呤的单链DNA，并精确识别脱氧腺苷到脱氧次黄嘌呤（dA-to-dI）的突变位点，从而阐明ABEs诱导的脱靶效应。通过涉及三种单向导RNA的研究，我们在原间隔区内外发现了许多意外的脱靶编辑。值得注意的是，ABE8e（V106W）表现出独特的脱靶特征，包括在以前未报道的位点（如RNF2和EMX1）有高编辑率（>10%）以及原间隔区外的突变。这些发现显著推进了我们对与ABEs相关的脱靶情况的理解。总之，我们的方法能够对ABE编辑组进行无偏分析，并为评估各种产生脱氧次黄嘌呤中间产物的新兴基因组编辑技术的特异性提供了一种广泛适用的工具。
