HEK-293 cells were cultured in DMEM + 10 %FBS, B cells were cultured in RPMI640 + 10%FBS. Plasmids were complexed with PEIpro in Opti-MEM and complexes were used to transfect 1x10^5 cells using a 24-well plate for CRISPR knock-in application.

Targeted DNA methylation is important for understanding transcriptional modulation and epigenetic diseases. Although CRISPR-Cas9 has potential for this purpose, it has not yet been successfully used to efficiently introduce DNA methylation and induce epigenetic diseases. We herein developed a new system that enables the replacement of an unmethylated promoter with a methylated promoter through microhomology-mediated end joining-based knock-in. We successfully introduced an approximately 100% DNA methylation ratio at the cancer-associated gene SP3 in HEK293 cells. Moreover, engineered SP3 promoter hypermethylation led to transcriptional suppression in human B lymphocytes and induced B-cell lymphoma. Our system provides a promising framework for targeted DNA methylation and cancer initiation through epimutations.