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Method for de novo Discovery of Locus Specific Regulatory Elements

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 1R43MH111294-01
Agency Tracking Number: R43MH111294
Amount: $225,000.00
Phase: Phase I
Program: SBIR
Solicitation Topic Code: 101
Solicitation Number: PA14-250
Solicitation Year: 2017
Award Year: 2016
Award Start Date (Proposal Award Date): 2016-09-05
Award End Date (Contract End Date): 2017-08-31
Small Business Information
Carlsbad, CA 92008-6509
United States
DUNS: 109145701
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 (323) 865-0846
Business Contact
Phone: (760) 431-1263
Research Institution

Project Summary
The advent of the CRISPR Cas 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 Cas 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 Cas dCas 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 enChIPandapos s usefulness for mental health research we
will target binding sites for Tcf l part of the Wnt Catenin pathway in E 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 we will establish
conditions for in vitro assembly of the sgRNA dCas complex and specific targeting with genomic DNA
Subsequent efforts will focus on determining optimal conditions for specific targeting of the sgRNA dCas RNP
complex with fixed chromatin extracted from cancer cells and primary mouse neurons Aim and mouse brain
tissues Aim 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 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 dCas 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

* Information listed above is at the time of submission. *

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