Targeted RNA <i>N</i>⁶‐Methyladenosine Demethylation Controls Cell Fate Transition in Human Pluripotent Stem Cells

Abstract

Abstract Deficiency of the N 6 ‐methyladenosine (m 6 A) methyltransferase complex results in global reduction of m 6 A abundance and defective cell development in embryonic stem cells (ESCs). However, it's unclear whether regional m 6 A methylation affects cell fate decisions due to the inability to modulate individual m 6 A modification in ESCs with precise temporal control. Here, a targeted RNA m 6 A erasure (TRME) system is developed to achieve site‐specific demethylation of RNAs in human ESCs (hESCs). TRME, in which a stably transfected, doxycycline‐inducible dCas13a is fused to the catalytic domain of ALKBH5, can precisely and reversibly demethylate the targeted m 6 A site of mRNA and increase mRNA stability with limited off‐target effects. It is further demonstrated that temporal m 6 A erasure on a single site of SOX2 is sufficient to control the differentiation of hESCs. This study provides a versatile toolbox to reveal the function of individual m 6 A modification in hESCs, enabling cell fate control studies at the epitranscriptional level.