Fig. 4: Evaluation of off-target editing activities of engineered Sdd7.

a Editing frequencies at potential off-target sites for BE4max, Sdd7, Sdd7e1, and Sdd7e2 were measured by targeted deep sequencing in HEK293T cells. PAM sequences are indicated in blue, and mismatched bases are shown in red lowercase letters. Specificity ratios were calculated by dividing the specificity of each Sdd7 variant (on-target frequency/off-target frequency) by that of the original Sdd7 (on-target frequency/off-target frequency). Data are presented as mean values, with error bars representing SEM from three independent biological replicates. b Editing frequencies at off-target sites captured by Digenome-seq for BE4max, Sdd7, Sdd7e1, and Sdd7e2 were quantified by targeted deep sequencing in HEK293T cells. Data are presented as mean ± SEM from three independent biological replicates. c OTI values for BE4max, Sdd7, Sdd7e1, and Sdd7e2 at the FANCF and HEK4 sites. The OTI is calculated as the ratio of the sum of base editing frequencies at all off-target sites to the on-target base editing frequency. Data are presented as mean values, with error bars representing SEM from three independent biological replicates. d Measurement of gRNA-independent off-target deamination activities of BE4max, Sdd7, Sdd7e1, and Sdd7e2 using the dSaCas9-mediated orthogonal R-loop assay in HEK293T cells. Data are presented as mean values, with error bars representing SEM from three independent biological replicates. e Representative heatmap depicting gRNA-independent off-target deamination frequencies measured by the dSaCas9-mediated orthogonal R-loop assay in HEK293T cells. f Cas9-independent RNA off-target deamination for BE4max, Sdd7, Sdd7e1, and Sdd7e2 in HEK293T cells was evaluated by transcriptome sequencing, which determined both the number of C-to-U edited nucleotides and the overall frequency of RNA C-to-U editing.