Targeted RNA N-6-Methyladenosine Demethylation Controls Cell Fate Transition in Human Pluripotent Stem Cells

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.