Assessment of the demethylation efficiency of Dem6A-Vec approach. (A) Targeted demethylation of the EEF2 target site (NM_001961.4 m⁶A:2886). Bar plots are used to demonstrate the normalized m⁶A abundance of the target site (EEF2 m⁶A:2886) and two nontarget sites, EEF2 m⁶A:2638 and RRAGA m⁶A:418. In addition, the relative expression of EEF2 mRNA is shown under actinomycin D treatment in a time line of 6 h to evaluate the mRNA stability of the target. (B) Targeted demethylation of the RRAGA target site (NM_006570.5 m⁶A:418). Bar plots are used to demonstrate the normalized m⁶A abundance of the target site (RRAGA m⁶A:418) and two nontarget sites, RRAGA m⁶A:1336 and EEF2 m⁶A:2886. In addition, the relative expression of RRAGA mRNA is shown under actinomycin D treatment in a time line of 6 h to evaluate the mRNA stability of the target. (C) Graphical demonstration of the normalized m⁶A abundance of both the EEF2 m⁶A:2886 and RRAGA m⁶A:418 sites for a 6 day posttransfection time line. (D) Normalized expression levels of the RfxCas13d mRNA expressed by Dem6A-Vec in the transfected cells for a 6 day posttransfection time line. The human housekeeping gene GAPDH was used for normalization purposes.