Targeted Manipulation of Cellular RNA m 6 A Methylation at the Single-Base Level.

Development of tools for precise manipulation of cellular mRNA m 6 A 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 m 6 A 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 m 6 A sites. The off-target effect was evaluated by whole transcriptome m 6 A 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 m 6 A methylation in a particular transcript.