Principal Investigators




    Contact information of lead PI



    Title of project or programme

    A CRISPR-Cas9 screen to identify genetic modifiers of APP/BACE-1 interactions

    Source of funding information

    NIH (NIA)

    Total sum awarded (Euro)


    Start date of award


    Total duration of award in years



    Acquired Cognitive Impairment... Aging... Alzheimer's Disease... Alzheimer's Disease including Alzheimer's Disease Related Dementias (AD/ADRD)... Biotechnology... Brain Disorders... Dementia... Genetics... Human Genome... Neurodegenerative... Neurosciences

    Research Abstract

    ? DESCRIPTION (provided by applicant): The overall goal of this proposal is to discover molecules along trafficking pathways leading to the convergence of two key proteins in Alzheimer’s disease (AD) pathogenesis – Amyloid Precursor Protein (APP) and ?-site APP-cleaving enzyme-1 (BACE-1). This convergence, and consequent enzymatic ?-cleavage of APP, is the rate-limiting step of amyloid beta (A?) production – a pathological hallmark of AD brains and a prevailing focus in AD research. Visualizing APP/BACE-1 trafficking in hippocampal neurons, we recently found that after synthesis, APP and BACE-1 are sorted into distinct vesicles, with BACE-1 selectively routed into recycling endosomes. At steady state, APP and BACE-1 convergence is a low-frequency event – producing A? at basal levels (Das et al., Neuron 2013; PMID: 23931995). Following up on these studies, we reasoned that ascertaining molecular pathways leading up-to this seminal convergence event would allow: 1) identification of the repertoire of trafficking pathways by which APP and BACE-1 meet to initiate the amyloidogenic cascade; and 2) discovery of novel “”druggable targets”” that can be manipulated to diminish APP/BACE-1 convergence and A? production. Towards this we developed an in-cellulo Optical assay to visualize Convergence of APP and BACE-1 (OptiCAB). Based on fluorescence complementation, this assay reports APP/BACE-1 interactions as a simple on/off readout, correlates with APP ?-cleavage, and is suitable for large-scale analyses. Combining this assay with a newly-developed powerful genome-scale screen using CRISPR-Cas9 knockout (GeCKO) library (collaboration with Feng Zhang, MIT), our goal is to discover genes involved in `trafficking-related’ upstream pathways that eventually lead to APP/BACE-1 convergence and A? production. Notably, CRISPR-Cas9- based screens are not limited by incomplete protein depletion and confounding off-target effects that have historically limited the utility of RNAi. Secondary validation of `hits’ (i.e. genes that attenuate APP/BACE-1 interactions) will be done in human induced pluripotent stem cells (iPSC’s); where APP-cleavage products will be analyzed after relevant CRISPR-knockout. Our aims are: Aim #1: Discover pathways leading to APP and BACE-1 convergence using OptiCAB and GeCKO; and Aim #2: Validate `hits’ from Aim 1 in human neuronally-differentiated iPSCs. Our experiments will not only provide insights into the physiologic “”amyloid-pathway”” in humans, but may also offer new targets for AD. Finally, note that our focus on the repertoire of trafficking pathways leading up-t APP/BACE-1 approximation stems from our own live-imaging studies; and is different from the current narrow focus on enzymatic activity of the secretases.

    Further information available at:

Types: Investments < €500k
Member States: United States of America
Diseases: N/A
Years: 2016
Database Categories: N/A
Database Tags: N/A

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