Figure 3.

Efficient editing of endogenous sites by dual-AAV split-PEs in human cells

(A) Schematic of split-PE delivered by dual-AAV1. N-terminal half and C-terminal half of split-PEs were packaged into two AAV1 separately, and then the cells were infected with both N-terminal half virus and C-terminal half virus, of which the ratio was 1:1. 5 days post-infection, cells were collected for sequencing. (B) Example of Sanger sequencing to detect editing at endogenous sites targeted by split-PEs. The red arrow indicates the edited site. (C) Targeted insertion of CTT at HEK3 in HEK293T cells. (D) Targeted transversion of G to T at VEGFA in HEK293T cells. (E) Targeted insertion of GTA at RNF2 in HEK293T cells. (F) Targeted insertion of GTA at RNF2 in HeLa cells. Editing efficiencies represent sequencing reads that contain the correct edit and do not contain indels among total sequencing reads, which were analyzed by MATLAB and CRISPResso2. Indels were plotted for comparison. AAV1-GFP, negative control. Values and error bars represent the mean ± SD of three independent biological repeats. One-way ANOVA was used for calculating statistical significance (∗∗p < 0.01; ∗∗∗p < 0.001).