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

Adenosine base editors (ABEs) facilitate the conversion of A•T base pairs to G•C base pairs, demonstrating significant potential for correcting pathogenic point mutations in humans. 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 dI-containing single-stranded DNA (ssDNA) and precisely identifies dA-to-dI mutation sites, thereby elucidating the off-target effects induced by ABEs. Through investigations involving three single-guide RNAs (sgRNAs), we identified numerous unexpected off-target edits both within and outside the protospacer regions. Analysis of these off-target sites revealed distinct characteristics of the ABE8e(V106W). These findings significantly advance our understanding of the off-target landscape associated with ABE8e. 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. Overall design: 10 samples are analyzed (6 sgRNA&ABE8e tranfacted sample, 2 ABE_only tranfated sample and 2 background sample) 20 samples are analyzed (8 sgRNA&ABE7.10 tranfacted sample in HEK293T, 4 sgRNA&ABE8e tranfacted sample in HEK293T, 4 sgRNA&ABE8e tranfacted sample in MCF7, 2 ABE8e_only tranfated sample in MCF7 and 2 background sample in MCF7)