Supplementary Figure 9: Side-by-side comparison of evoCas9, SpCas9-HF1 and eSpCas9(1.1) specificity on selected therapeutically relevant genes
From: A highly specific SpCas9 variant is identified by in vivo screening in yeast
293T cells were transfected with wild-type SpCas9, SpCas9-HF1, eSpCas9(1.1) or evoCas9 together with sgRNAs targeting the FANCF2 (a) or the CCR5 loci (b). Mean indel formation at the on-targets and at two previously validated off-target sites (one for each locus) was evaluated 7 days post-transfection using the TIDE tool. The sequences of the on- and off-target sites for each locus are reported above the corresponding graphs; the red square indicates the mismatched base. (c) On/off ratios calculated from the mean indel percentages obtained in (a). The dotted lines indicate evoCas9 fold increase in specificity. (d) Schematic representation of the CCR5 locus and its off-target site in the highly homologous CCR2 gene. Simultaneous cleavage of the two sites generates a chromosomal deletion of approximately 16 kb. Semi-quantitative PCR was performed on genomic DNA of 293T cells transfected with wild-type SpCas9, SpCas9-HF1, eSpCas9(1.1) or evoCas9 and the CCR5 sgRNA to assess the amount of chromosomal deletion generated in each condition. The FANCF locus was used as an internal normalizer. The amount of deletion was quantified using densitometry with ImageJ. The numbers above the dotted line indicates evoCas9 fold increase in specificity relative to the connected histogram bars. For all panels n=2 biologically independent samples (represented as overlaid circles).
