Dem6A-Vec, a versatile type VI CRISPR-based approach for targeted m6A demethylation in mRNAs

The aim of this study was to develop, optimize, and validate a versatile tool for site-specific m6A demethylation in mRNAs. To this end, we present Dem6A-Vec, an in-house developed 'all-in-one' plasmid vector designed to enable targeted demethylation of m6A sites in human mRNAs. In contrast to previous approaches, Dem6A-Vec supports the simultaneous expression of a guide RNA (gRNA) specific to the mRNA site of interest and a catalytically inactive Cas13d fused to the major m6A eraser ALKBH5 (dCas13d-ALKBH5). The presented approach enhances flexibility and efficiency by allowing rapid targeting of diverse m6A-modified mRNA transcripts through a simplified gRNA replacement workflow. Dem6A-Vec is intended to facilitate investigations into the functional roles of m6A modifications at defined transcriptomic sites in mammalian cells.