DNA Epigenome Editing Using Crispr-Cas Suntag-Directed DNMT3A

DNA methylation, an epigenetic modification, has widespread effects on gene expression during development. However, our ability to assign specific function to regions of DNA methylation is limited by the poor correlation between global patterns of DNA methylation and gene expression. To overcome this barrier, we utilized nuclease-deactivated Cas9 protein fused to repetitive peptide epitopes (SunTag) recruiting multiple copies of antibody-fused de novo DNA methyltranferase 3A (DNMT3A) (CRISPR-Cas SunTag-directed DNMT3A) to amplify local DNMT3A concentration and to methylate genomic sites of interest. Here, we demonstrated that CRISPR-Cas SunTag-directed DNMT3A not only dramatically increased CpG methylation but also, to our surprise, CpH (H =A or C or T) methylation at the HOXA5 lociin human embryonic kidney 293T cells (HEK293T). Furthermore, using a single sgRNA, CRISPR-Cas SunTag-directed DNMT3A was capable of methylating 4.5 kb genomic regions, surpassing previous targeted methylation tools whose activity is limited to 200bp. Using reduced representation bisulfite sequencing (RRBS) and RNA-seq, we concluded that CRISPR-Cas SunTag-directed DNMT3A methylated regions of interest without affecting global DNA methylome and transcriptome. This effective and precise tool enables site-specific manipulation of DNA methylation and may be used to address the relationship beteween DNA methylation and gene expression.