The FMR1 Promoter Was Methylated Using A Site-directed Epigenetic Modification Technique

Fragile X syndrome is a commonly inherited form of developmental delay,due to the mutation at FMR1 gene.CGG repeats within the 5' untranslated region of the FMR1 gene expand from an average normal allele length of 30 triplets to well over 200 repeats in patients with fragile X syndrome.At the threshold of about 200 repeats,designated as the “full mutation”,the CpG island of FMR1 promoter becomes heavily methylated and the chromotin status shifts from a euchromatic state to a fully heterochromatic state.These epigenetic abnormalities lead to the transcriptional silencing of FMR1 and the absence of FMRP(fragile X mental retardation protein).However,certain characteristics like the hypermethylation of the FMR1 locus seen in FXS patients failed to be recapitulated by non-human model system,making it necessary to establish system that models epigenetic abnormalities in human cells.Currently most disease models are generated by conditional gene knocking out,such as the Loxp/Cre system,and gene editing technologies including ZFN?TALEN and CRISPR/Cas9 system.There is no disease model established using epigenome editing technology.We combined the Casilio(Cas9+Pumilio)system and DNMT enzymes,exploring whether we could specifically methylate the promoter of FMR1 to reduce its transcription in hESC,establihsing a model of FXS.The most parts of this research are focused on revealing whether the Casilio-DNMT plasmids and the screened guide RNA targeting FMR1 promoter could work efficiently.We have performed a serise of transfections in 293 T cell and then harvestd those cells followed by RT-PCR and BSP analysis.In addition,we have constructed the lentivirus plasmids to generate a cell line expressing Casilio DNMT3A-DNMT3 L stablely.Now we have identified efficient sgRNAs and confirmed that Casilio DNMT3A-DNMT3 L system could direct site-specific DNA methylation at the FMR1 promoter in 293 T cell.In addition,we have generated hESC lines that stablely expressing Casilio DNMT3A-DNMT3 L.