RESUMEN
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.
Asunto(s)
Metilación de ADN , ADN de Cadena Simple/genética , Epigénesis Genética , Edición Génica/métodos , Proteínas de Neoplasias/genética , Factor de Transcripción Sp3/genética , Secuencia de Bases , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Células Clonales , Citosina/metabolismo , ADN de Cadena Simple/metabolismo , Técnicas de Sustitución del Gen , Genoma , Células HEK293 , Humanos , Proteínas de Neoplasias/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Factor de Transcripción Sp3/metabolismo , Transcripción GenéticaRESUMEN
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.
