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Method for de novo Discovery of Locus Specific Regulatory Elements

Award Number: R43MH111294
ORGANIZATION: NATIONAL INSTITUTE OF MENTAL HEALTH
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/05/2016
PERIOD OF PERFORMANCE END DATE: 08/31/2017

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Method for de novo Discovery of Locus Specific Regulatory Elements - Project Summary 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/ß-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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2018 ( Subtotal = $0 )
2018	2016	ACTIVE MOTIF, INC.	1914 PALOMAR OAKS STE 150	CARLSBAD	CA	92008	SAN DIEGO	USA	Mental Health Research Grants	000	1	1/16/2018	NEW	$0
														Subtotal = $0

Issue Date FY: 2016 ( Subtotal = $225,000 )
2016	2016	ACTIVE MOTIFF LLC	1914 PALOMAR OAKS WAY, STE 150	CARLSBAD	CA	92008	SAN DIEGO	USA	Mental Health Research Grants	000	1	9/5/2016	NEW	$225,000
														Subtotal = $225,000

Grand Total All Awards = $225,000

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Method for de novo Discovery of Locus Specific Regulatory Elements

Award Number: R43MH111294

ORGANIZATION: NATIONAL INSTITUTE OF MENTAL HEALTH

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 09/05/2016

PERIOD OF PERFORMANCE END DATE: 08/31/2017

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