Fig. 6: en31-ABEmax8.17d enables robust and flexible base editing in human cells.

a Schematic showing the architecture of all-in-one plasmids expressing adenine base editors as T2A-EGFP fusion with gRNA cloning sites. b Schematic showing the mode of the action of an adenine base editor (ABE). The nick position by ABE is indicated by a scissor on the nick strand. Target base is on the edit strand. c Schematic showing the architecture of the gamma-globin promoter highlighting the point mutations responsible for Hereditary persistence of fetal hemoglobin (HPFH) condition. Two clusters of point mutations located at 200 and 115 nucleotides from the transcription start site (TSS) are zoomed in. d, e Ballon plots showing the A to G editing events (%) as obtained from amplicon sequencing upon targeting -113/-116 (d) and -111 (e) sites of HBG1/2 promoter by FnCas9-ABEmax8.17d, en31-ABEmax8.17d, and SpNG-ABEmax8.17d with sgRNA containing 20/21-nt protospacer (g20 and g21 respectively) in HEK293T cells. Target bases for adenine base editing are highlighted in red and numbered w.r.t. g21. f, g Line plot showing A to G editing events (%) plotted on the Y-axis obtained from amplicon sequencing upon targeting endogenous loci having alternately present target ‘A’ bases by en31-ABEmax8.17d in HEK293T cells. Target bases (As) for adenine base editing are numbered on the X-axis and marked red. Error bars represent mean ± SEM of n = 3 independent biological replicates. h Schematic showing the adenine base editing window of en31-ABEmax8.17d. Apex of the bell curve indicates the optimal editing window. Numbers indicate the base positions across the protospacer of the gRNA. i Schematic showing the comparative base editing window between Sp-ABEmax8.17d (shaded in pink) and en31-ABEmax8.17d (shaded in green). j Balloon plot showing modulation of the base editing window by en31-ABEmax8.17d with gRNAs of varying spacer lengths (g21−26) at EMX1 locus in HEK293T cells. k Schematic showing the sliding of the base editing window from primary window (shown in red) to secondary window (shown in blue) by x-/sx-gRNAs. d, e, j Untransfected cells were used as control. Values represent the mean of n = 3 independent biological replicates. Source data are provided as a Source Data file.