Extended Data Fig. 7: Additional data and analysis of the on-target DNA and off-target RNA activities of BE3 and SECURE BE3 variants.

a, Dot plots illustrating on-target DNA editing efficiencies of nCas9–UGI–NLS (control), wild-type BE3, BE3-R33A and BE3-R33A/K34A in HEK293T cells on 12 genomic sites. These are the same data as shown in Fig. 2c, expanded to include all cytosines across the spacer sequence. b, Jitter plots from RNA-seq experiments in HepG2 cells showing RNA cytosines modified by wild-type BE3, BE3-R33A and BE3-R33A/K34A. Data for wild-type BE3 are from the experiments presented in Fig. 1c (replicates 2–4). n, total number of modified cytosines identified. c, Manhattan plots of data showing the distribution of modified cytosines induced with BE3-R33A or BE3-R33A/K34A expression for replicate 3 from b overlaid on modified cytosines induced with wild-type BE3 expression (the wild-type BE3 data are the same in the top and bottom plots). n, total number of modified cytosines identified. d, Heat map of on-target DNA base editing efficiencies of nCas9–UGI–NLS (control), wild-type BE3, BE3-R33A and BE3-R33A/K34A in HepG2 cells with the RNF2 gRNA (cells from same experiment as shown in b). Replicates 1, 2 and 3 for wild-type BE3 and nCas9–UGI–NLS show the same data presented as replicates 2, 3 and 4 for wild-type BE3 and nCas9–UGI–NLS in Fig. 1b. Bases within the editing window of the on-target spacer sequence are numbered as previously described. Note again the inclusion of position C12. e, Dot plot for HepG2 on-target data shown in d, expanded to include all cytosines across the spacer sequence. f, Schematic of the editing windows (coloured boxes) for wild-type BE3, BE3-R33A and BE3-R33A/K34A based on experimental data from Fig. 2c and Extended Data Fig. 7a. Darker-coloured and more-translucent boxes indicate positions generally showing higher and lower C-to-T editing efficiencies, respectively. Increased stringency for a 5′T with BE3-R33A/K34A is also indicated. The PAM (NGG) and the nicking site in the DNA backbone are highlighted. Drawings are adapted with permission from table 1 of ref. ¹.