Whitehead Institute for Biomedical Research and Department of Biology, MIT, Cambridge, MA 02124, USA
The development of the iPS cell technology has revolutionized our ability to study development and diseases in defined in vitro cell culture systems. The talk will focus on the use of gene editing for the study of epigenetic regulation in development and disease.
1. Monitoring the dynamics of DNA methylation at single cell resolution during development and disease: DNA methylation is a broadly studied epigenetic modification that is essential for normal mammalian development. Current methods to quantify methylation provide only a static "snap shot" of DNA methylation, thus precluding the study of real-time methylation dynamics during cell fate changes. We have established a new approach that enables monitoring loci-specific DNA methylation dynamics at single-cell resolution.
2. Editing DNA methylation in the mammalian genome: The functional significance of specific methylation events in development and disease remains elusive due to lack of experimental approaches to edit these events. We developed a DNA methylation editing toolbox that fusion of either the catalytic domain of Tet1 or Dnmt3a protein to a catalytic inactive Cas9 (dCas9) to achieve targeted DNA methylation editing with co-expression of target-specific guide RNAs. (i) We first validated this tool by turning on or off two endogenous methylation reporters. (ii) With application of dCas9-Tet1, we observed that active de-methylation can be induced in BDNF promoter IV to activate its expression in mouse cortical neurons, and showed (iii) that de-methylation of MyoD distal enhancer facilitates reprogramming fibroblasts into myoblasts and subsequent myotube formation. (iv) We show that dCas9-Dnmt3a can open CTCF-mediated chromatin loops by targeted methylation of specified CTCF anchor site thus blocking its binding. Our results established that a modified CRISPR system with dCas9 fused by DNA modification enzymes can be assembled into DNA methylation editing tools to study the functional significance of specific methylation event in the mammalian genome. (v) Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.