The advent of the CRISPR/Cas9 technology has revolutionized genome engineering resulting in a vast array of new applications based on the ability of a single guide RNA (sgRNA) to target a Cas9 protein in living cells. Engineered DNA-binding molecule-mediated chromatin immunoprecipitation, or enChIP, was developed and patented by the Fujii lab at Osaka University, and uses transient transfection of an sgRNA and an epitope- tagged deactivated Cas9 (dCas9) to target a specific genomic region. This approach allows for the identification of DNA and proteins that interact with a specific genomic region without prior information as to what those interacting components are, and thus provides a comprehensive understanding of locus specific genomic regulation. The need for transfection limits enChIP to cells that can be grown and transfected in culture. One of the challenges that plagues neuroscience research is the lack of cell culture models that accurately replicate neurological states. To demonstrate enChIP's usefulness for mental health research we will target binding sites for Tcf7l2, part of the Wnt/??Catenin pathway, in E18 mouse neurons and tissue. The experiments described in this Phase I SBIR proposal will establish proof of concept that enChIP can be adopted for use in a cell-free system, which will enable its broad use for neuroscience research thereby increasing our understanding of genomic regulation in the nervous system. In Aim 1 we will establish conditions for in vitro assembly of the sgRNA/dCas9 complex and specific targeting with genomic DNA. Subsequent efforts will focus on determining optimal conditions for specific targeting of the sgRNA/dCas9 RNP complex with fixed chromatin extracted from cancer cells and primary mouse neurons (Aim 2) and mouse brain tissues (Aim 3). Success of these Phase I efforts will result in the development of methods for the identification of proteins and DNA looping events at the targeted locus and will form the basis of future Phase II efforts, which will expand to include identification of locus-associated regulatory RNAs and the expansion of the technology to other cell and tissue types. We envision that the commercial potential of cell-free enChIP will be first be realized initially as a custom service and eventually as an assay kit consisting of reagents and a detailed protocol for the neuroscience research community and for the life sciences field in general.
Public Health Relevance Statement: Active Motif Project Narrative enChIP allows the unprecedented isolation of a single genomic locus along with any co-bound proteins and nucleic acids, but requires transfection of targeting guideRNA//dCas9 constructs. Here, we propose the adaptation of enChIP to work on chromatin isolated from any cell type without the need for transfection and transient expression of the targeting constructs and expands the potential of this technology to neurobiological disease models. Identifying novel proteins, their isoforms or mutations and DNA looping events that are specific to a diseased state will offer unique therapeutic targets.
Project Terms: Acceleration; Adopted; Affect; Animal Model; Antibodies; base; beta catenin; Binding Sites; Biological; Biological Assay; Biological Sciences; Boxing; Brain; brain tissue; cancer cell; CCCTC-binding factor; Cell Culture Techniques; Cell Survival; cell type; Cell-Free System; Cells; Chromatin; chromatin immunoprecipitation; Chromatin Loop; Chromatin Structure; Co-Immunoprecipitations; Communities; community living; Complex; CRISPR/Cas technology; crosslink; Custom; Data; design; Disease model; DNA; DNA Binding; DNA-Binding Proteins; Engineering; Epitopes; Event; experience; Future; Gene Expression Regulation; Generations; Genes; Genome engineering; Genomic DNA; Genomic Segment; Genomics; Guide RNA; HCT116 Cells; Histocompatibility Testing; Immunoprecipitation; In Vitro; in vitro testing; Large Intestine Carcinoma; Legal patent; Life; Mass Spectrum Analysis; Mediating; Mental Health; method development; Methods; Modeling; Mus; Mutation; nervous system disorder; Nervous system structure; Neurobiology; Neurologic; Neurons; Neurosciences; Neurosciences Research; novel; nucleic acid binding protein; Nucleic Acids; Pathway interactions; Phase; Plague; Preparation; Protein Isoforms; Proteins; Protocols documentation; Publishing; Reagent; Recombinants; Regulation; Regulatory Element; Research; research study; RNA; sample fixation; Services; Small Business Innovation Research Grant; success; TCF7L2 gene; Technology; Testing; therapeutic target; Tissue Fixation; tissue fixing; Tissues; tool; Transfection; Universities; Work
