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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. eLife, 11 . Art. No. e81398. ISSN 2050-084X. PMCID PMC9578707. doi:10.7554/elife.81398.

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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’) or 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 are not optimized for extracellular interactions. Cell-based screening is a promising technology for the deorphanization of 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 transmembrane (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 assays. The newly deorphanized ligands include three receptor protein tyrosine phosphatase (RPTP) ligands and a chemokine-like protein that binds to killer immunoglobulin-like receptors (KIRs). These new interactions provide a resource for future investigations of interactions between the human-secreted and membrane proteomes.

Item Type:Article
Related URLs:
URLURL TypeDescription ItemDiscussion Paper CentralArticle
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 authors were supported by The Howard Hughes Medical Institute (KCG), the G Harold and Leila Y Mathers Charitable Foundation (KCG), and NIH 1RO1-GM150125 (KCG and KZ).
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
G. Harold and Leila Y. Mathers Charitable FoundationUNSPECIFIED
PubMed Central ID:PMC9578707
Record Number:CaltechAUTHORS:20221110-429809700.6
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:117822
Deposited By: Research Services Depository
Deposited On:27 Nov 2022 20:23
Last Modified:28 Nov 2022 17:28

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