Extended Data Figure 9. Targeted m⁶A demethylation controls expression of Mycn in paused ESCs.

a. Model of lentiviral dCasRx epitranscriptomic editor with the m⁶A demethylase ALKBH5 and single guide RNA. b. Validation of the expression of the dCasRx-ALKBH5 fusion by western blot in parental (non-infected) ESCs, infected ESCs, and 2 infected clones. Clone 5 was used for all experiments (representative of 2 biological replicates). c. Guide RNAs (gRNAs) transfected for non-targeting control and Mycn-targeting conditions. d. Changes in m⁶A using the dCasRx-ALKBH5 editor in paused ESCs. Guides #2 and #3 significantly reduce m⁶A in Mycn transcripts, as measured by m⁶A-qPCR, and were selected for all subsequent experiments. N=7 biological replicates. NT: non-targeting. e. Dot blot showing that the global increase of m⁶A in paused ESCs is not affected by the dCasRx-ALKBH5 editor, with Mettl3^−/− ESCs as negative control. Representative of 3 biological replicates. MB: methylene blue. f. Quantification of N-Myc protein levels, showing increased expression with the dCasRx-ALKBH5 editor targeting Mycn in paused ESCs, with representative blot shown in Fig. 6e. N=3 biological replicates. g. Demethylation of Mycn increases the total RNA levels per cell in paused ESCs (n=6 biological replicates).

Data are mean ± SD (d, f-g) and P-values (as indicated on figure) by one-way ANOVA with Dunnett’s multiple comparison tests (d, f-g).