Locus-Specific Regulation of Expression Using the CRISPR-Cas9-Based System.

DNA methylation inhibitor or loss and gain of function of DNA methylation key players were widely used to investigate the regulation of X inactive-specific transcript () expression by DNA methylation, which results in global change of DNA methylation. Here, we reported a novel method for regulation of using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. First, expression was increased in 5-aza-2'-deoxycytidine-treated female goat fibroblast cells. Second, three single-guide RNAs (sgRNAs) that target the differential methylation region (DMR) were inserted to deactivated Cas9 (dCas9) nuclease and the catalytic domain of the DNA methyltransferase Dnmt3a coexpression plasmid. Bisulfite PCR analysis and quantitative real-time PCR revealed that the methylation level of the DMR was significantly increased, while the expression of was downregulated in all three sgRNAs, compared with the mock-transfected cells. Third, the methylation activity at the sites of 37 bp from the protospacer-adjacent motif sequence showed the strong change relative to the mock-transfected cells. Furthermore, genome-wide DNA methylation and expression of the DNA methylation key players were not statistically changed in all three sgRNAs. Therefore, we confirmed that expression was regulated by DNA methylation, and directed DNA methylation of DMR at locus-specific solution decreased expression.