Homo sapiens Raw sequence reads of IminiBEs and SIminiBEs

IscB is likely the ancestor of CRISPR Cas9 that shares a similar domain organization. IscB nuclease or nickase could be harnessed for genome editing. Particularly the small size of IscB satisfies the packaging capacity of adeno-associated virus (AAV) as a vector for gene therapy. The base editing efficiency of OgueIscB-RNA in mammalian cells is less than 5% on average, which makes it difficult to use for gene therapy. Here, we developed high-efficiency miniature base editors by engineering OgueIscB and its cognate RNA, named IminiBEs. We demonstrated the robust C-to-T conversion by IminiCBEs and A-to-G conversion by IminiABEs. The editing efficiency of C-to-G by IminiCGBE and A-to-Y by IminiAYBE is relatively low, less than 20% on average. Fusing non-specific dsDNA binding protein Sso7d to the N-terminus of IscB can increase BE efficiency 1.2-3 folds, and we termed it SIminiBEs. We validated the effective target-adjacent motif (TAM) of engineered OgueIscB in mammalian cells to be NNRR, which is less restricted than the NGG PAM of SpCas9. In sum, IminiBEs and SIminiBEs are flexible, efficient and conven-ient base editors, which expand the toolbox for genome editing and gene therapy.