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. 2021 Feb 5;49(4):2390–2399. doi: 10.1093/nar/gkab052

Figure 3.

Figure 3.

Romidepsin improves base editing efficiency by affecting the protein and gRNA expression levels and the chromatin state. (A) Flow cytometry analysis of the expression levels of ABE7.10–2A-EGFP, spCas9–2A-EGFP and BE3–2A-EGFP proteins in the presence and absence of romidepsin. The proportion of GFP-positive cells increased following romidepsin treatment. (B) Western blot assay to demonstrate the ABE7.10 protein expression levels in HEK293T/17 cells. The expression levels of ABE7.10 proteins increased in the presence of romidepsin. GAPDH is shown as a loading control. (C) Flow cytometry analysis of the expression levels of CMV, EFS, and hPGK promoter-driven ABE7.10-2A-EGFP proteins in the presence and absence of romidepsin. Romidepsin improved both EFS and hPGK promoter-driven protein expression. (D) Targeted deep sequencing analysis showed that romidepsin improved the base editing efficiency at both CCR5 and RNF2 sites in HEK293T/17 cells transfected with EFS- and hPGK promoter-driven ABE7.10 expressing plasmids. (E) Quantitative real-time PCR analysis for detecting gRNA expression levels. Romidepsin increased U6 promoter-driven gRNA expression levels. (F) Base editing efficiencies at four endogenous target sites in RNP delivered HEK293T/17 cells. (G) ChIP assay results at four endogenous target sites. The acetylation percentage at the four sites increased with romidepsin treatment. Error bars indicate SEM (n = 3); ns, not significant; P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (using two-tailed Student's t-test).