Using Fto Functional Analysis And Single-base Editing Technology To Study The Role Of M~6A Demethylation

N6-methyladenosine(m~6A)is the most abundant modification in eukaryotic mRNA.The dynamic and reversible modification of m~6A is conducted by methyltransferase and demethylases,which determines the fate of m~6A-modified mRNA,playing a vital role in regulating gene expression,protein translation,cell behaviors,and physiological conditions.Fto is a kind of m~6A demethylase,and its biological function is unknown in brain development.Therefore,we constructed Fto knockout mice(Fto-KO)and explored the biological role of m~6A demethylation in mouse brain development by analyzing the Fto function.The results showed that,fourteen days after birth,the body weight,body length and brain weight of Fto-KO mice were significantly lower than those of wild-type mice.Secondly,the whole brain size of Fto-KO mice significantly decreased at P14,especially the cerebellum was serious malformation.In addition,Nissl staining result showed that the thickness of cerebral cortex in Fto-KO mice was significantly thinned.These phenomenons indicate that the knock-out of Fto causes abnormal brain development in mice,suggesting that m~6A demethylation plays an important role in regulation of brain development.In addition,there is no single m~6A site demethylation function study.We attempted to establish a single m~6A site demethylated cell model using the newly reported single-base editing techniques(xCas9(3.7)-ABE(7.10)and CBE4-Gam systems,reported by David Liu).In 293T cells,we mutated m~6A sites of four genes(BSG,TUG1,MALAT1 and ACTB),respectively.The results showed that we successfully edited the m~6A site in ACTB gene,which laid the foundation for studying function of this m~6A demethylation.In conclusion,we found that m~6A demethylation has an important effect on mouse brain development.In addition,we successfully mutated the m~6A site of ACTB gene.This provides a new way to study the m~6A demethylation.