Selict-seq profiles genome-wide off-target effects in adenosine base editing

Abstract

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.