Fig. 1

High-specificity Cas9 variants with attenuated cleavage activity. a Schematics of Cas9-WT, engineered Cas9 variants, and sgRNA variants. Locations of the alanine substitutions introduced in Cas9-WT to create eCas9-1.1 or Cas9-HF1 are shown with blue or red asterisks, respectively. Red triangles indicate cleavage positions. GX₁₉ sgRNA starts with a G matched to its protospacer (blue line). gX₁₉ sgRNA has a mismatched G at its 5’-end. gX₂₀ sgRNA includes an extra guanine at its 5’ terminus. The protospacer-adjacent motif (PAM) is shown by a red line labeled NGG. H, not G (A or C or T); D, not C (A or G or T). b, c Western blot (b) and indel frequencies at the EMX1 site (c) of HeLa cells co-transfected with plasmids encoding Cas9-WT or variants and EMX1-specific sgRNA. Cas9 variants were expressed by new plasmids used in this study or by old plasmids used in previous studies. Error bars, s.e.m of two or three biological replicates