Supplementary Figure 1: Examination of APOBEC3’s effect on the base substitution frequency of sgRNA and ODNs.

(a) Endogenous expression of APOBEC-AID family members in 293FT and HeLa cells. mRNA levels of APOBEC-AID family members were determined by RT-qPCR and normalized against TATA binding protein (TBP) mRNA levels. (b) Western blots of the whole-cell lysates from wild type 293FT cells (293FT) and 293FT cells stably expressing the C-terminal HA tagged APOBEC3B (A3B) or the catalytically inactive one (A3Bm). Western blots were performed using anti-HA antibody. Alpha tubulin served as loading control. Uncropped blot images are shown in Supplementary Data Set 1. The blots shown are representative of three experiments. (c) Relative APOBEC3B mRNA level in 293FT and A3B cells. APOBEC3B mRNA levels were determined by RT-qPCR and normalized against TATA binding protein (TBP) mRNA levels. (d) Schematic diagram illustrating the procedures to examine the base substitutions in the sgRNAs transfected into cells. (e) Base substitution frequency of each base in the spacer regions of indicated sgRNAs that are either non-transfected or transfected into 293FT or A3B cells. The data were from two independent experiments. (f) Base substitution frequency of each base in ssODNs or dsODNs that are transfected into A3B or A3Bm cells. (g) Comparison of the theoretical base substitution fractions at each dinucleotide calculated from the base content of ssODNs and the experimentally determined ones in 293FT and A3B cells. The base substations on C of CpC or TpC reflected APOBEC3 mutational signatures. Red arrows highlight the same base substitutions as observed in the ODN-cognate genomic regions in Fig. 2. (a, c, f) The means (±s.d.) were from three independent experiments.