Targeted DNA methylation in vivo using an engineered dCas9-MQ1 fusion protein (RRBS)

Comprehensive studies have shown that DNA methylation plays vital roles in both loss of pluripotency and governance of the transcriptome during embryogenesis and subsequent developmental processes. Aberrant DNA methylation patterns have been widely observed in tumorigenesis, aging, and neurodegenerative diseases, highlighting the importance of a systematic understanding of DNA methylation and the dynamic changes of methylomes during disease onset and progression. Here we describe a facile and convenient approach for efficient targeted DNA methylation by fusing inactive Cas9 (dCas9) with an engineered prokaryotic DNA methyltransferase MQ1. Our study presents a lentivirus-free strategy to achieve locus-specific cytosine modifications in the genome within 24 hours. Finally, we demonstrated our tool can induce targeted CpG methylation in mice by zygote microinjection, thereby facilitating the dissection of the functional relevance of specific CpG methylation marks at endogenous loci. I) ChIP-seq of plasmid electroporated K562 cells using Illumina Nextseq sequencing system. II) Reduced representation bisulfited sequencing (RRBS) of plasmid transfected HEK 293T cells using Illumina Hiseq2000 sequencing system. III) PCR amplicon deep sequencing of HEK 293T cells, K562 cells or mice tissue using Illumina Nextseq sequencing system.