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Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform

Siepe, Dirk H. and Henneberg, Lukas T. and Wilson, Steven C. and Hess, Gaelen T. and Bassik, Michael C. and Zinn, Kai and Garcia, K. Christopher (2022) Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform. . (Unpublished)

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Secreted proteins, which include cytokines, hormones and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell-surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors (“orphans”) and are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening do not work well for extracellular interactions. Cell-based screening is a promising technology for definition of new ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell-surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding. The newly deorphanized ligands include three receptor tyrosine phosphatase (RPTP) ligands and a chemokine like protein that binds to killer cell inhibitory receptors (KIR’s). These new interactions provide a resource for future investigations of interactions between the human secreted and membrane proteomes.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Discussion Paper ItemJournal Article
Siepe, Dirk H.0000-0002-0009-8023
Henneberg, Lukas T.0000-0003-3477-4541
Hess, Gaelen T.0000-0002-3836-0277
Bassik, Michael C.0000-0001-5185-8427
Zinn, Kai0000-0002-6706-5605
Garcia, K. Christopher0000-0001-9273-0278
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. The authors thank the Howard Hughes Medical Institute (K.C.G.), and the G. Harold and Leila Y. Mathers Charitable Foundation (K.C.G.). Author Contributions Conceptualization, K.C.G., D.H.S.; Methodology, D.H.S., M.C.B. and K.G.G.; Library Design: D.H.S., K.Z., G.T.S., M.C.B. and K.C.G.; Screening, D.H.S., L.T.H.; Validation, D.H.S., S.C.W.; Analysis, D.H.S.; Investigation, D.H.S., L.T.H., and K.C.G.; Data Curation, D.H.S., K.Z.; Writing – Original Draft, D.H.S. and K.Z.; Writing – Review & Editing, K.C.G., D.H.S, and K.Z.; Visualization, D.H.S.; Supervision, K.C.G.; Funding Acquisition, K.C.G. The authors have declared no competing interest.
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
G. Harold and Leila Y. Mathers Charitable FoundationUNSPECIFIED
Subject Keywords:CRISPRa, pooled library, Cell-surface, protein-protein interaction, screen, receptor, ligand, protein communities, secreted proteome
Record Number:CaltechAUTHORS:20220706-965608000
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Official Citation:Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform Dirk H. Siepe, Lukas T. Henneberg, Steven C. Wilson, Gaelen T. Hess, Michael C. Bassik, Kai Zinn, K. Christopher Garcia bioRxiv 2022.06.22.497261; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:115355
Deposited By: George Porter
Deposited On:08 Jul 2022 22:40
Last Modified:23 Nov 2022 22:03

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