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  1. Evaluation Summary:

    This manuscript reports the development of a proteo-genomic screening methodology to identify protein-protein interactions between secreted proteins and their cell surface receptors. The authors use a CRISPRa-based approach to overexpress membrane proteins in cells and then use magnetic cell sorting to identify receptors that bind candidate ligands. This approach leads to the identification of several novel interaction pairs that are validated biochemically, including receptor tyrosine phosphatase ligands and other interactions with implications for immune system function. The work is of interest to a wide variety of fields including biochemistry and signaling.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    In this manuscript, Siepe et al. developed a high-throughput screen designed to identify novel protein-protein interactions in the extracellular human proteome. Their CRISPRa-based method induced the expression of transmembrane receptors such that they could be screened for binding to proteins of interest. Major strengths of this approach include the ability to screen multiple ligands in parallel, the ability to identify low-affinity interactions, and the availability of custom single- and multi-pass transmembrane protein libraries for selective target screening. A potential weakness is that low-affinity binders and non-specific interactions can be difficult to distinguish in certain cases, and these scenarios require more complex statistical analysis. The authors also note that the CRISPRa strategy cannot induce the expression of multi-subunit receptors that may be required for some ligands. The screen was tested against a curated set of ligand candidates and identified more than twenty novel interactions with intriguing biological implications. Both the method and newly discovered interactions will be of immediate scientific interest given the growing need to identify receptors for orphan ligands. Overall, this technology should function as a powerful new tool for ligand deorphanization in the extracellular space.

  3. Reviewer #2 (Public Review):

    The authors implemented a novel interaction screening strategy where binders to soluble bait are identified by selecting/sorting cells expressing a library of putative receptors, followed by sequencing of selected cells to identify receptors. The expression library is created by CRISPR activation. The method's shortcomings are explained well in the article (at the end) which need not be repeated here. The new set of interactions discovered/validated are surprising and interesting, which may lead to the opening of the relevant fields to new avenues of thought and research. While co-expression data hint at vague biological functions, none are tested, which is beyond the scope of the manuscript. I have no major concerns about the methodology, which appears to be executed well.

  4. Reviewer #3 (Public Review):

    The authors use a previously developed technology, CRISPR activation screening, in which pooled sgRNAs are used to guide an RNA-associated regulatory complex (MS2-p65-HSF1 transcriptional activators) to promoter regions resulting in increased expression of a specific target gene. The authors screen two different pooled libraries TM1 (single pass) and TM2+ (multiple pass) with 20 different recombinant biotinylated soluble ligands and identified 22 novel interactions. These interactions were further characterized by SPR and cell-based binding experiments; however, several of the interactions are low affinity and were not characterized for any activity or function beyond the relatively weak biochemical binding. Therefore, while the data provide evidence of potential novel interactions, the biological relevance remains unclear.