Fig. 3: Off-target assessment of AID-2S, −3S and rAPOBEC1 base editors.
From: Cytosine base editing systems with minimized off-target effect and molecular size
a Targeted RNA sequencing of three gRNA-independent locus reported before (CTNNB1, RSL1D1, and IP90) was performed for four rAPOBEC1 CBEs and three Target-AID CBEs. Data are presented as mean values ± sd (n = 3). Statistically significant differences between BE4max and other CBEs were supported by two-tailed unpaired Student’s t-test; p-value (P* = 1.98E-03 ~ 2.36E-03 for RSL1D1 site, 1.45E-02 ~ 2.18E-02 for IP90 site, 8.90E-03 ~ 1.33E-02 for CTNNB1 site). The gRNA-dependent RNA off-target was also shown in Supplementary Fig. S7. b Evaluation of Cas9-dependent off-target effects. The two HEK2 off-target sites and four VEGFA off-target sites were analyzed by DNA amplicon deep sequencing. AID-2S, -3S showed significant reduction compared to Target-AID at HEK2 site 1 and VEGFA site 2, 3, and 4, supported by the two-tailed unpaired Student’s t-test; p-value (P* = 1.79E-06 ~ 9.88E-04 for AID2S, P** = 6.02E-06 ~ 8.13E-04 for AID3S). The mutation frequencies at each nucleotide position are shown in Supplementary Fig. 8. Data are presented as mean values ± sd (n = 3). c gRNA-independent DNA off-target was compared by using seven off-target R-loop sites (see Methods for detail). Compared to Target-AID, AID-2S and -3S showed significant reduction across all sites, supported by two-tailed unpaired Student’s t-test; p-value (P* = 3.36E-06 ~ 5.37E-03 for AID2S, P** = 2.89E-06 ~ 8.93E-04 for AID3S). Inlaid_1247_YE1 also showed a significant difference to YE1 at site 5, 6 and 7 (P*** = 4.59E-02, 7.59E-04, 1.59E-04). Off-target frequencies are shown as the percentage of reads containing the mutation. Data are presented as mean values ± sd (n > =3). The mutation frequencies at each nucleotide position are also shown in Supplementary Fig. 9. d On-target editing versus average off-target editing profile for all CBEs tested in this study. The y axis represents the mean on-target editing at the eight on-target, and the x axis represents the mean off-target editing at the seven orthogonal R-loop sites. Source data are provided as a Source Data file.
