Figure 3.

Nexturastat A significantly boosted BE3-mediated gene editing in mouse embryos and correction of ABCA4 mutation. (A) Summary of the viability of blastocysts from zygotes injected with BE3 mRNA and different concentration of eGFP-sgRNA, and treated with Nexturastat A (0.5 μM). (B) Fluorescence images of blastocysts with the editing. Scale bar, 100 μm. (C) NGS results showed C to T conversion rate in obtained embryos at eGFP locus (p.Q183X). Total embryos from each group were randomly divided into three groups. (D) A schematic of BE3-NG-mediated gene correction of ABCA4 (CAT to CGT missense mutation). The target base is shown in green, and the PAM is shown in blue. (E) Sanger sequencing results of the colonies showed that the mutant ABCA4 (mABCA4) gene was corrected by BE3-NG. The mutated and corrected nucleotide is highlighted with red star. (F) The C-to-T base editing frequencies at ABCA-site 1 and ABCA-site 2. mABCA4, pCAG represents partial human ABCA4 fragments harboring mutation and CAG promoter, respectively. Error bars, S.E.M.; n = 3; Control, DMSO treated group; ns, not significant; **P < 0.01, ***P < 0.001, ****P < 0.0001.