Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein

Studies on biological functions of N-6-methyladenosine (m(6)A) modification in mRNA have drawn significant attention in recent years. Here we describe the construction and characterization of a CRISPR-Cas13b-based tool for targeted demethylation of specific mRNA. A fusion protein, named dm(6)ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5-125 (dPspCas13b) to m(6)A demethylase AlkB homolog 5 (ALKBH5). dm(6)ACRISPR specifically demethylates m(6)A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate beta-catenin-encoding CTNNB1 mRNA that contains multiple m(6)A sites to trigger its translation. In addition, the dm(6)ACRISPR system incurs efficient demethylation of targeted epitranscriptome transcripts with limited off-target effects. Targeted demethylation of transcripts coding for oncoproteins such as epidermal growth factor receptor (EGFR) and MYC can suppress proliferation of cancer cells. Together, we provide a programmable and in vivo manipulation tool to study mRNA modification of specific genes and their related biological functions.