High-throughput engineering of combinatorial chromatin signals and epigenetic cellular memory

PROJECT SUMMARY/ABSTRACT A major frontier of genomic medicine is to convert annotations of disease-associated variants and gene expression states into actionable therapeutic targets. Genomic sequence editing by CRISPR/Cas has garnered heightened interest as potential therapeutics, but still carries risks of off-target mutagenicity, and is less effective at targeting cis-regulatory elements, where the majority of disease-associated variants reside. A conceptual alternative to DNA sequence editing is to deliver a brief pulse of a synthetic therapeutic that can trigger memorized silencing of target promoters and enhancers in a "hit-and-run" strategy, bypassing concerns of mutagenicity associated with DNA sequence editing, and immunogenicity associated with constitutive expression of CRISPR/Cas components. In practice, engineering sustained epigenetic transcriptional silencing has had mixed success, but recent efforts demonstrate that constructing specific combinations of chromatin signals -- broadly encompassing cis- regulatory elements, regulatory protein complexes, and covalent chromatin modifications -- can be critical for success. However, existing tools for manipulating chromatin signals at high throughput are limited in combinatorial capacity. This proposal builds the knowledge base and generalizable CRISPR/Cas tools to overcome such limitations to enable systematic engineering of epigenetic silencing memory across genomic loci in human cells. The results will pave the way for future epigenetic therapeutics that expand the directly targetable portion of the human genome, including cis-regulatory elements that can be highly specific with respect to cell types and disease pathophysiology. To achieve these objectives, I am proposing to train in CRISPR/Cas synthetic biology, functional genomics, single-cell methods and neurogenetics. This training will be mentored by a co-mentorhsip team consisting of Dr. Luke Gilbert (UCSF, Innovative Genomics Institute, and Arc Institute), Dr. Howard Chang (Stanford) and Dr. Thomas Montine (Stanford), each providing critical intellectual and physical resources for specific areas proposed in the training plan and my career development. My career goal is to conduct research at the forefront of genome regulation and genomic medicine as an academic principle investigator.