DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A [RNA-Seq]. Homo sapiens

We demonstrate that dCas9-SunTag-DNMT3A dramatically increased CpG methylation at the HOXA5 locus in human embryonic kidney 293T cells (HEK293T). Furthermore, using a single sgRNA, dCas9-SunTag-DNMT3A was able to methylate a 4.5 kb genomic region and repress HOXA5 gene expression. Reduced representation bisulfite sequencing (RRBS) and RNA-seq showed that dCas9-SunTag-DNMT3A methylated regions of interest with minimal impact on the global DNA methylome and transcriptome. Overall design: I)PCR amplicon deep sequencing of dCas9-SunTag-DNMT3A treated HEK2937 samples using Illumina Nextseq sequencing system. II) Reduced representation bisulfited sequencing (RRBS) of plasmid transfected HEK 293T cells using Illumina Hiseq2000 sequencing system. III) Whole genome bisulfite sequencing of dCas9-SunTag-DNMT3A treated HEK2937 samples. IV) RNA sequencing of dCas9-SunTag-DNMT3A treated HEK2937 samples

本研究证实，dCas9-SunTag-DNMT3A可显著提升人胚肾293T细胞（human embryonic kidney 293T，HEK293T）中HOXA5基因座的CpG甲基化水平。此外，仅需单条单向导RNA（single guide RNA，sgRNA），dCas9-SunTag-DNMT3A即可对4.5 kb的基因组区域进行甲基化修饰，并抑制HOXA5基因的表达。简化代表性亚硫酸氢盐测序（Reduced Representation Bisulfite Sequencing，RRBS）与RNA测序（RNA-seq）结果显示，dCas9-SunTag-DNMT3A仅靶向目标区域进行甲基化，对全局DNA甲基组与转录组的影响极小。

整体实验设计如下：
I) 采用Illumina Nextseq测序平台，对经dCas9-SunTag-DNMT3A处理的HEK2937样本开展PCR扩增子深度测序；
II) 采用Illumina Hiseq2000测序平台，对质粒转染的HEK293T细胞开展简化代表性亚硫酸氢盐测序（RRBS）；
III) 对经dCas9-SunTag-DNMT3A处理的HEK2937样本开展全基因组亚硫酸氢盐测序；
IV) 对经dCas9-SunTag-DNMT3A处理的HEK2937样本开展RNA测序。