Editing CG and non-CG DNA methylation to identify genomic elements that regulate gene expression

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PROJECT SUMMARY / ABSTRACT A long-standing goal in biology is to define the relationship between genotype and phenotype. A major surprise of the human genome project was that the human genome encodes so few genes despite the complexity of cell types that compose for example, the human brain. As such it is assumed that combinatorial gene expression programs are key for specifying the function of specialized cell types such as neurons. Cell type specific gene expression programs therefore must be encoded by cis- and trans- non-coding regulatory DNA elements whose function is regulated by the epigenetic code and key proteins such as transcription factors. Elucidating how non-coding regulatory elements function to program cells will transform our understanding of human biology, development and disease. CRISPR/dCas9 technologies enable us to move beyond correlative studies, by editing the epigenome and determining the direct effect of epigenetic alterations on gene expression. We have created a new epigenetic editing functional genomics approach that we have named CRISPRoff. CRISPRoff robustly and specifically writes CpG DNA methylation (5mC) and repressive histone modifications to target loci. We are proposing to use CRISPRoff to map all genomic regulatory elements that are regulated by 5mC across an entire human chromosome. In the proposed experiments we will use perturb-seq, which combines pooled CRISPR screens with a single cell transcriptome readout, to directly measure how deposition of 5mC by CRISPRoff across an entire chromosome modulates gene expression. This approach will identify genetic regulatory elements key for induced pluripotent stem cells and neurons, a key step to understanding how tissue-specific gene expression is controlled. Our proposed research will serve to demonstrate the utility of this approach and motivate extending this approach to map gene regulatory elements across the entire human genome. The results of the proposed research will serve as a fundamental resource and roadmap for a broad community of biomedical scientists and greatly inform our understanding of human biology and disease.