Figure 2. Editing efficiency of the Target-AID base editor at Y402H CFH variant (rs1061170). A: Sanger sequencing chromatograms showing the target locus. Control refers to HEK293A-CFH(p.His402) cells that have been treated with a combination of the Target-AID base editor with a sgRNA targeting LacZ. The second chromatogram displays results following transfection with the Target-AID base editor and a sgRNA specific to the rs1061170 region. The highlighted region is the putative activity window of Target-AID showing the incidence of a C:G to T:A nucleotide correction. B: Base calling for targeted next-generation deep sequencing of the base-editor treated HEK293A-CFH(p.His402) cells. Percentages represent the fraction of sequencing reads occupied by a particular nucleotide at the specified position. The target protospacer is highlighted in red and each nucleotide is numbered 1 to 20 in which the PAM consensus sequence is considered positions +21 to +23. Deep sequencing shows that 21.5% of the target cytosines were edited into thymine nucleotides at position C1. Additionally, the percentage of indel formation is 0.15% (amplicon results of three independent biologic replicates). C: List of sgRNA sequences screened at the top 11 off-target sites that also contain at least one cytosine in the putative Target-AID activity window in the presence of a NGG PAM site. Each off-target site was evaluated using next generation deep sequencing and no off-target base editing was identified.