Tailoring and reversing m6A editing with sequential RNA bioorthogonal chemistry

Many existing methods for post-transcriptional RNA modification rely on a single-step approach, limiting the ability to reversibly control m6A methylation at specific sites. Here, we address this challenge by developing a multi-step system that builds on the concept of sequential RNA bioorthogonal chemistry. Our strategy uses an azide-based reagent (NAI-N3) capable of both cleavage and ligation reactions, thereby allowing iterative and reversible modifications of RNA in living cells. By applying this approach in CRISPR (clustered regularly interspaced short palindromic repeats)-based frameworks, we demonstrate tailored editing of m6A marks at targeted RNA sites, overcoming the one-way restriction of conventional bioorthogonal methods. This sequential protocol not only broadens the scope for fine-tuned RNA regulation but also provides a versatile platform for exploring dynamic m6A function in genetic and epigenetic research.