Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion.

Cell reports methods 2023 May 22

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Cellular differentiation requires global changes to DNA methylation (DNAme), where it functions to regulate transcription factor, chromatin remodeling activity, and genome interpretation. Here, we describe a simple DNAme engineering approach in pluripotent stem cells (PSCs) that stably extends DNAme across target CpG islands (CGIs). Integration of synthetic CpG-free single-stranded DNA (ssDNA) induces a target CpG island methylation response (CIMR) in multiple PSC lines, Nt2d1 embryonal carcinoma cells, and mouse PSCs but not in highly methylated CpG island hypermethylator phenotype (CIMP)+ cancer lines. MLH1 CIMR DNAme spanned the CGI, was precisely maintained through cellular differentiation, suppressed MLH1 expression, and sensitized derived cardiomyocytes and thymic epithelial cells to cisplatin. Guidelines for CIMR editing are provided, and initial CIMR DNAme is characterized at TP53 and ONECUT1 CGIs. Collectively, this resource facilitates CpG island DNAme engineering in pluripotency and the genesis of novel epigenetic models of development and disease. © 2023 The Author(s).

Citation

Joshua Tompkins, Elizabeth Lizhar, Alireza Shokrani, Xiwei Wu, Jordan Berley, Diba Kamali, Deborah Hussey, Jonas Cerneckis, Tae Hyuk Kang, Jinhui Wang, Walter Tsark, Defu Zeng, Swetha Godatha, Rama Natarajan, Arthur Riggs. Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion. Cell reports methods. 2023 May 22;3(5):100465