Deficiency of the N -methyladenosine (m A) methyltransferase complex results in global reduction of m A abundance and defective cell development in embryonic stem cells (ESCs). However, it's unclear whether regional m A methylation affects cell fate decisions due to the inability to modulate individual m A modification in ESCs with precise temporal control. Here, a targeted RNA m 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 A site of mRNA and increase mRNA stability with limited off-target effects. It is further demonstrated that temporal m 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 A modification in hESCs, enabling cell fate control studies at the epitranscriptional level.
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| http://dx.doi.org/10.1002/advs.202003902 | DOI Listing |
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