Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells

DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate their roles and genomic targets. Disruption of DNMT3A or DNMT3B individually, as well as of both enzymes in tandem, creates viable, pluripotent cell lines with distinct effects on their DNA methylation landscape as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome the immediate lethality, we generated a doxycycline (DOX) responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1 mutant lines. However, DOX-mediated repression of the exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT mutant ESCs, including single base genome-wide maps of their targets.