Fig. 2: ATR is essential for knock-in, regardless of the donor vector type, and its activation enhances the knock-in efficiency.

a Western blotting illustrating that the ATR inhibitor (10 µM VE821) effectively inhibits ATR activity in genome-edited cells. Protein was harvested 12 h post-genome editing. b Genome-editing results with a plasmid donor vector following treatment with two ATR inhibitors (10 µM VE821 and 1 μM ceralasertib). The ATR inhibitors significantly decreased the efficiencies of both knock-in and EJ-TI, underscoring the role of ATR in targeted insertion with the plasmid donor vector. Data are presented as mean ± SD. (n = 3 independent experiments). Dunnett’s multiple comparison tests were conducted. c Genome editing results with an AAV donor vector following treatment with ATR inhibitors. The decreased efficiencies of the knock-in underscore the role of ATR in targeted insertion with the AAV donor vector. Data are presented as mean ± SD. (n = 3 independent experiments). Dunnett’s multiple comparison tests were conducted. d Schematic representation of the hypothesis to modulate ATR activity through the KEAP1 inhibitor. e Western blotting illustrating the upregulation of NRF2 following treatment with a KEAP1 inhibitor (300 µM 4-Octyl Itaconate (4OI)) in cells with IR-induced (6 Gy) DNA damage. A modest increase in phosphorylated ATR was observed. To prevent IR-induced oxidation, the cells were treated with 5 mM N-acetyl-L-cysteine (NAC). Protein extraction was performed 8 h after exposure. f Genome editing results with a plasmid donor vector following treatment with a KEAP1 inhibitor (300 μM 4OI) in reporter ES cells. The KEAP1 inhibitor increased the efficiencies of knock-in and EJ-TI. Data are presented as mean ± SD. (n = 3 independent experiments). Unpaired t-tests were conducted. g Genome editing results with an AAV donor vector following treatment with a KEAP1 inhibitor (300 μM 4OI) in reporter ES cells. The KEAP1 inhibitor significantly increased the knock-in efficiency. Data are presented as mean ± SD. (n = 3 independent replicates). Unpaired t-tests were conducted.