Project Summary/Abstract Almost every cell in a human being has the same DNA sequence yet different cells vary widely in which genes are expressed. In addition to the genetic code, human cells contain chemical modifications on DNA called epigenetics that can dictate cell type identity and cell fate. The most abundant epigenetic modification in the human genome is DNA methylation, which is critical for silencing transposable elements, genomic imprinting, and X chromosome inactivation. Aberrant DNA methylation patterns is a hallmark of aging and cancer cells, however the genetic pathways that contribute to DNA methylation remains unclear. My research laboratory aims to unlock how DNA methylation is established in the human genome and to understand how defects in DNA methylation pathways leads to disease. Using genome-wide genetic screens, my lab has recently discovered genes that previously have not been implicated in DNA methylation, including an RNA binding protein and the proteasomal complex that degrades proteins. In the next five years, we will dissect the roles of our newly discovered genetic factors of DNA methylation and how they regulate gene expression programs in cancer and embryonic stem cells using multidisciplinary approaches including biochemistry, genetics, and advances in DNA sequencing technologies. In addition to our goals to understand DNA methylation pathways, my laboratory is a leader in pioneering new CRISPR methods to write and erase DNA methylation at any site in the genome. We use lessons learned from our basic biology findings to engineer tools that allow us to turn off/on human genes solely by changing the epigenetics of a gene instead of inducing harmful DNA breaks. In the next five years, we will innovate next generation CRISPR epigenetic editing technologies that enables fine tuning of transcription at defined levels. To overcome the current challenge of delivering CRISPR technologies into primary human cells, we will establish the first protein-based delivery of CRISPR epigenetic editing technologies into primary immune cells for use in cancer immunotherapy applications. Our CRISPR epigenetic editing technologies and delivery platforms will have broad use in the biomedical sciences and as safe and robust tools for cell and gene therapy clinical applications purely by modifying epigenetics instead of genome editing.