Supplementary Figure 8: APOBEC3 binds to genomic ssDNA regions exposed near Cas9 nickase-generated SSBs and induces indel formation.

(a) Indel frequencies induced by Cas9-generated DSB at indicated activation time points in primary human T cells that are pretreated with control siRNA (siCtrl) or siRNA against endogenous APOBECs (siA3(Mix)). (b) Normalized indel frequencies (D10A- or dCas9-induced indel frequencies relative to Cas9-induced ones) at indicated activation time points in primary human T cells. The indel frequencies induced by Cas9 increased during T cell activation (a), suggesting that the sgRNA-Cas9 RNP electroporation efficiency may increase during T cell activation. Therefore, D10A-induced indel frequencies (Fig. 3f) were normalized against Cas9-induced ones to exclude the effect of electroporation efficiency. (c) APOBEC3 knockdown suppressed the indel formation induced by Cas9 nickase-generated SSB in episomal shuttle vector. (d) A3B cells were either non-transfected (NT) or co-transfected with indicated sgRNAs and Cas9, D10A, H840A or dCas9. ChIP-qPCR assays were then performed to detect the binding capacities of histone H3 (using H3 antibody, H3 ab) at indicated genomic loci. The results from the anti-6×His-tag antibody (Ctrl ab) were included as negative controls. The means (±s.d.) were from three independent experiments. (a-c) The data were from two independent experiments.