Effect Of Specific Modification The DNA Methylation At CTCF Binding Sites Of H19/IGF2 Locus On Mouse Embryonic Stem Cells And Early Embryo

Genomic imprinting is an epigenetic mechanism for controlling imprinted gene expression,regulating an unequal expression of the maternally or paternally derived alleles at a specific locus,genomic imprinting modulates growth and development in mammals and is associated with genetic disorders.In human,closely linked H19 and Igf2 are located on chromosome 11p15.5,shares a set of enhancer.The imprinted monoallelic expression is controlled by differential methylation of the H19/Igf2 intergenic differentially methylated region(DMR),in which the DNA methylation patterns affect the binding of CCCTC-binding factor(CTCF).So far,the binding site for CTCF and its DNA methylation status need to be elucidated.In this study,CRISPR/dCas9-mediated DNMT3 A or TET were specially designed to target CTCF binding sites for modifying the DNA methylation status,their effct on the expression of imprinted gene and the development of preimplantation embryo were discussed.In this study,eight gRNA were designed according to the four binding sites for the CCCTC binding factor(CCCTC-binding factor,CTCF)at the H19/Igf2 gene locus,and a pgRNA-humanized-Igf2 vector were constructed and co-transfected with CRISPR/dCas9-Dnmt3 a or CRISPR/dCas9-Tet into MEFs cells.Results show that the gRNA targeted the third CTCF binding site had a more obvious effect on the expression of Igf2 gene.When gRNA were co-transfected with CRISPR/dCas9-Dnmt3 a into MEFs cells,the expression of Igf2 gene were increased,the DNA methylation in the promoter region were increased which detected by Bisulfite PCR sequencing,in addition,results showed that the proliferation of MEFs cells was accelerated which detected by CCK8 method,and the cell cycle of MEFs cells was shortened;When gRNA were co-transfected with CRISPR/dCas9-Tet,the expression of Igf2 gene were decreased,the DNA methylation in the promoter region were decreased,cell proliferation were slowed down,and the cell cycle was prolonged.After co-transfection,the expression of a panel of genes related to Igf2 genes was examined,the results showed that the expression of Igf1 r,Irs1,Akt1 and Mapk1 genes was positively correlated with the expression of Igf2.After gRNA-guided CRISPR/dCas9-Dnmt3 a was transferred into mouse embryonic stem cell,the results showed that the methylation of the targeted site was significantly increased,and the expression of Igf2 was increased,while the expression of pluripotential factors Nanog and Oct4 genes was not affected.To study the effect of targeted modifying DNA methylation on mouse early embryonic development,gRNA and CRISPR/dCas9-Dnmt3 a was co-injected into the cytoplasm of fertilized eggs,results showed that the expression of Igf2 was significantly increased,the development rate of 2-Cell and 4-Cell embryos was increased,and the embryo development was accelerated.The expression of Igf1 and Igf2 r were decreaed,while Igf1 r,Akt1,Mapk1,Irs1,Rps6 and mTOR were increased.In conclusion,CRISPR/dCas9 can be used to modify the DNA methylation status of the CTCF binding sites at H19 / Igf2 gene locus,and the expression of Igf2 gene and Igf2-related genes were affected,while the pluripotential genes were not affected.After co-injected into the cytoplasm of fertilized eggs,dCas9-Dnmt3 a can significantly improve the Igf2 expression,and increase the development rate and accelerate embryo development.This study laid a foundation for further elucidated the regulation of imprinting genes.