Supplementary Figure 4: APOBEC3 introduces sparse base substitutions in genomic DNA near Cas9 nickase-generated SSBs.

(a) Schematic diagram illustrating the procedures to determine the base substitution and indel mutations induced by Cas9 variant-generated breaks in genomic DNA. (b) Base substitution frequency induced by Cas9 variant-generated breaks in genomic DNA. 293FT cells were either left non-transfected (NT) or co-transfected with indicated sgRNAs and Cas9, D10A, H840A or dCas9. Base substitution frequency of each base in the upstream and downstream 25-bp region of Cas9 cleavage sites was measured by deep sequencing. Red arrow indicates the APOBEC3-featured base substitution. Asterisk indicates a base substitution specifically induced by sgVEGFA-3-Cas9, which however was not up-regulated by APOBEC3B over-expression (data not shown). Other mutations were all background base substitutions that remain largely unchanged in all tested situations (compare Cas9, D10A, H840A, dCas9 and NT with each other). (c) Base substitution frequency induced by sgVEGFA-D10A-generated SSB was up-regulated by A3B overexpression. 293FT, A3B or A3Bm cells were co-transfected with sgVEGFA together with Cas9, D10A, H840A or dCas9, respectively. (b, c) The means (±s.d.) were from three independent experiments.