Fig. 1
RCas9-methyltransferase like 3 (METTL3) system enhances N6-methyladenosine (m6A) modification. a Upper panel: a schematic representation of the deactivated Cas9 (dCas9) and METTL3 catalytic domain (METTL3CD) fusion proteins. Lower panel: a modified single guide (sg) RNA construct with puromycin. b Methylated RNA immunoprecipitation (MeRIP) assays showing the abundance of m6A of CUB domain-containing protein 1 (CDCP1) mRNA in SV-HUC-1 cells expressing RCas9-M3. c A schematic diagram of the ligation-amplification method for single-base m6A validation. d Validation results of four different sites. The positions of the m6A sites (155, 173, and 212) were numbered relative to the first nucleotide of the 3′ untranslated region (UTR). The three sites were confirmed as m6A sites, whereas the other site was unmodified and treated as a control. e dCas9-M3 nuclear co-export with CDCP1 mRNA. The dCas9-M3 was delivered to SV-HUC-1 cells with a sgRNA and short protospacer adjacent motif-containing ssDNA molecule (PAMmer) targeting the 3′ UTR of CDCP1. Scale bars: 10 μm. Blue pseudocolor: 4′,6-diamidino-2-phenylindole staining of the nuclei. f Fraction of cells with a nuclear signal. Mean values ± standard error of the mean (SEM; n = 40). g, h RIP assays of enhanced green fluorescent protein (EGFP) after stable transfection of the RCas9-M3 system in SV-HUC-1 cells targeting CDCP1 mRNA compared to non-targeting sgRNA and PAMmer or EGFP alone. g Real-time quantitative polymerase chain reaction (qRT-PCR) analysis of RCas9-M3 RIP in SV-HUC-1 cells with CDCP1 3′ UTR primers. h. Western blot of EGFP. Data are presented as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001