Figure 2

Base editing of human PCSK9 in vitro. (A) Codons encoding glutamine, arginine, or tryptophan residues that could potentially be changed to stop codons with C-to-T or G-to-A edits. (B) On-target base editing was assessed by Sanger sequencing for various codons in human PCSK9 in HEK 293T cells using BE3, BE3-VQR, or BE3-VRER. The protospacer and PAM sequences for each target site are listed, with the bold letters corresponding to the editing “window” (13 to 17 bases upstream of the PAM) and the underline indicating the target codon (either sense or antisense), in which a C-to-T change would result in a stop codon on the sense strand. “–” indicates no base editing detected, “+” indicates modest base editing, and “++” indicates substantial base editing. (C) Top left panel: CEL-I nuclease assays were performed with genomic DNA from HEK 293T cells transfected with BE3 and a guide RNA targeting the codon encoding Q302. Arrows show the cleavage products resulting from the CEL-I nuclease assays; the intensity of the cleavage product bands relative to the uncleaved product band corresponds to the mutagenesis rate. MW = molecular weight. Top right panel and bottom panels: Sanger sequencing confirmed base editing (C-to-T) of Q302 (codon CAG) in both HEK 293T cells and induced pluripotent stem cells.