CellFIE: CRISPR- and Cell Fusion-based Two-hybrid Interaction Mapping of Endogenous Proteins |
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| Affiliation: | 1. Neuroproteomics, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany;2. Department of Neurology, Charité Universitätsmedizin Berlin, Berlin, Germany;3. Brain Development and Disease, Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria;1. State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China;2. University of Chinese Academy of Sciences, Beijing 100049, China;3. Signalling Research Centers BIOSS and CIBSS, University of Freiburg, Freiburg, Germany;4. Department of Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany;5. Centre for Chronic Immunodeficiency (CCI), University Clinics and University of Freiburg, Freiburg, Germany;6. National Facility for Protein Science in Shanghai, ZhangJiang Lab, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201203, China;7. School of Pharmacy, Tianjin Medical University, Tianjin 300070, China;1. Laboratory of Biological Chemistry, Department of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece;2. Division of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Athens, Greece;3. Institute of Biosciences, University Research Center of Ioannina, Ioannina, Greece;1. Center for Network Systems Biology, Boston University, Boston, MA, USA;2. Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA;3. Department of Biology, Boston University, Boston, MA, USA |
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| Abstract: | Numerous genetic methods facilitate the detection of binary protein–protein interactions (PPIs) by exogenous overexpression, which can lead to false results. Here, we describe CellFIE, a CRISPR- and cell fusion-based PPI detection method, which enables the mapping of interactions between endogenously tagged two-hybrid proteins. We demonstrate the specificity and reproducibility of CellFIE in a matrix mapping approach, validating the interactions of VCP with ASPL and UBXD1, and the self-interaction of TDP-43 under endogenous conditions. Furthermore, we show that CellFIE can be used to quantify changes of endogenous PPIs upon stress induction or drug treatment. For the first time, CellFIE facilitates systematic mapping of interactions between endogenously tagged proteins and represents a novel tool to characterize PPIs in live cells under dynamic conditions. |
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| Keywords: | protein–protein interactions (PPIs) endogenous tagging two-hybrid BRET cell fusion |
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