Targeted Manipulation of Cellular RNA m⁶A Methylation at the Single-Base Level

Development of tools for precise manipulation of cellular mRNA m⁶A methylation at the base level is highly required. Here, we report an RNA-guided RNA modification strategy using a fusion protein containing deactivated nuclease Cas13b and m⁶A methyltransferase METTL14, namely, dCas13b-M14, which is designedly positioned in the cytoplasm. dCas13b-M14 naturally heterodimerizes with endogenous METTL3 to form a catalytic complex to methylate specific cytoplasmic mRNA under a guide RNA (gRNA). We developed assays to screen and validate the guiding specificity of varied gRNAs at single-base resolution. With an optimum combination of dCas13b-M14 and gRNAs inside cells, we have successfully tuned methylation levels of several selected mRNA m⁶A sites. The off-target effect was evaluated by whole transcriptome m⁶A sequencing, and a very minor perturbation on the methylome was revealed. Finally, we successfully utilized the editing tool to achieve de novo methylations on five selected mRNA sites. Together, this study paves the way for studying position-dependent roles of m⁶A methylation in a particular transcript.