Fig. 1: Off-target analysis of DdCBE for mitochondrial and nuclear genome editing with GOTI.

a GOTI workflow for analyzing off-target profile of DdCBE. b The DdCBE target for generating the m.G12918A point mutation (D393N), m.C12336T nonsense mutation (Q199stop), and m.G12341A silent mutation (Q200Q) in the ND5 protein. Translation triplets are underlined and target sequences with possible editing loci are shown in red. c On-target efficiency of ND5-DdCBE (m.G12918A and m.C12336T) for tdTomato⁺ and tdTomato^– cells on the basis of WGS. Distribution pattern of off-target sites in m.G12918A (d) and m.C12336T (e) E14.5 fetuses (red dots) with Cre fetuses as control (blue dots). Spacer represents region between recognition sequences of the TALE pair. Each dotted box indicates a single off-target event. f Comparison of the total number of identified off-target SNVs in Cre and ND5-DdCBE (m.G12918A and m.C12336T) injected groups by WGS. g Number of indels identified in Cre and ND5-DdCBE (m.G12918A and m.C12336T) injected groups by WGS. h Proportion of C·G to T·A mutations among all identified SNVs for Cre and ND5-DdCBE (m.G12918A and m.C12336T) injected groups. i Distribution of mutation types. The number in each cell indicates the proportion of a certain type of mutation among all mutations. j The distribution of off-target SNVs in the transcribed and untranscribed regions. k SNVs identified from all DdCBE-edited samples did not overlap, suggesting that off-targets on the nuclear genome were mainly caused by the sequence-independent activity of DdCBE. l Sequence logos generated from off-target sequences with C·G to T·A conversions by ND5-DdCBE in nuclear genome. Bits reflect sequence conservation at a given position. Data are presented as the means ± SEM. P values were evaluated with the unpaired Student’s t-test (two-tailed).