Editing of DNA methylation using CRISPR/Cas9 and a ssDNA template in human cells

Programmable DNA methylation is required for understanding of transcriptional regulation and elucidating gene functions. We previously reported that MMEJ-based promoter replacement enabled targeted DNA methylation in human cells. ssDNA-mediated knock-in has recently been reported to completely reduce random integrations. We speculated that by changing MMEJ-to ssDNA-based knock-in, targeted DNA methylation may be achieved through a hemimethylation-symmetric methylation pathway. We herein successfully developed a new system that enables the replacement of an unmethylated promoter with a methylated ssDNA promoter through ssDNA-based knock-in. A DNA methylation ratio of approximately 100% was achieved at the cancer-associated gene SP3 in HEK293 cells. The present results provide a promising framework for artificial epigenetic modifications. •Programmable DNA methylation is required for understanding transcriptional regulation and elucidating gene functions.•We previously reported that MMEJ-based promoter replacement enabled targeted DNA methylation in human cells.•ssDNA-mediated knock-in has recently been reported to completely reduce random integrations.•We successfully introduced a DNA methylation ratio of approximately 100% at the cancer-associated gene SP3 in HEK293 cells using ssDNA-based knock-in.•Engineered SP3 promoter hypermethylation led to transcriptional suppression.