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

Abstract

Deficiency of the N⁶ -methyladenosine (m⁶ A) methyltransferase complex results in global reduction of m⁶ A abundance and defective cell development in embryonic stem cells (ESCs). However, it's unclear whether regional m⁶ A methylation affects cell fate decisions due to the inability to modulate individual m⁶ A modification in ESCs with precise temporal control. Here, a targeted RNA m⁶ 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 m⁶ A site of mRNA and increase mRNA stability with limited off-target effects. It is further demonstrated that temporal m⁶ 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 m⁶ A modification in hESCs, enabling cell fate control studies at the epitranscriptional level.