Journal: Molecular Therapy. Nucleic Acids
Article Title: Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities
doi: 10.1016/j.omtn.2022.07.026
Figure Lengend Snippet: Construction of the hESC colony with inducible CE-8e-dV expression (A) Relative A-to-G base-editing efficiencies of the four editors in SHhES8 cells at three sites that already tested in HEK293T cells (n = 3 independent replicates). No significant differences were observed between any of two pairs as determined by one-way ANOVA. (B) Comparison of the editing efficiency of the four editors at three sites by deep sequencing in HEK293T cells and SHhES8 cells. Each dot represents the editing efficiency of each edited base (n = 3 independent replicates). The error bars represent the standard error of the mean (SEM) values. ns, not significant, p > 0.05, Student’s t test. (C) Schematic diagram for constructions of TRE3Gs-CE-ABE8e (V106W + delta 153)-RY and PB-CAG-Tet-On 3G-IRES2-Hyg, which are inserted into the SHhES8 cell line. (D) Representative images of SHhES8 cells with long-term stable CE-8e-dV expression in the absence or presence of doxycycline (dox). Scale bars represent 100 μm. (E) Comparison of the A-to-G base-editing efficiencies between the induction system (+dox) and transfection system (−dox with transfection) at 12 sites in the CE-8e-dV stable SHhES8 cell line. Each dot represents the highest editing efficiency (n = 3 independent replicates).
Article Snippet: For inducible ABE long expression in hESCs, the TRE3Gs fragment was synthesized by GenScript and cloned into a piggyBac plasmid containing the 5′ and 3′ PiggyBac homology arms.
Techniques: Expressing, Comparison, Sequencing, Transfection
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