Targeted RNA m6A Editing Using Engineered CRISPR-Cas9 Conjugates

Abstract

N⁶-methyladenosine (m⁶A) is a major epitranscriptomic mark exerting crucial diverse roles in RNA metabolisms, including RNA stability, mRNA translation, and RNA structural rearrangement. m⁶A modifications at different RNA regions may have distinct molecular effects. Here, we describe a CRISPR-Cas9-based approach that enables targeted m⁶A addition or removal on endogenous RNA molecules without altering the nucleotide sequence. By fusing a catalytically inactive Cas9 with engineered m⁶A modification enzymes, the programmable m⁶A editors are capable of achieving RNA methylation and demethylation at desired sites, facilitating dissection of regional effects of m⁶A and diversifying the toolkits for RNA manipulation.

Keywords: CRISPR-Cas9; Demethylation; Methylation; N6-methyladenosine; RNA targeting.